/•'rout  i 


STUDIES  IN  IMMUNITY 


BY 

PROFESSOR  JULES    BORDET 

PROFESSOR   OF   BACTERIOLOGY   AT   THE   UNIVERSITY   OF   BRUSSELS 
DIRECTOR    OF   THE    PASTEUR  INSTITUTE   OF   BRABANT 

AND   HIS   COLLABORATORS 


COLLECTED  AND  TRANSLATED 

BY 

FREDERICK   P.  GAY,  A.B.,  M.D. 

INSTRUCTOR    IN    PATHOLOGY,     HABVAKD    MEDICAL    SCHOOL 


INCLUDING  A  CHAPTER  WRITTEN  EXPRESSLY  FOR  THIS 
PUBLICATION    BY    PROFESSOR    BORDET 


FI R 'S  ?   E  D 1  2' I  tf  if 

FIRST    THOUSAND 


NEW  YORK 

JOHN  WILEY  &  SONS 

LONDON.-   CHAPMAN  &  HALL,  LIMITED 

1909 


COPYRIGHT,  1909,  BY 
FREDERICK  P.  GAY 


„  .1  "•••*  '•••  '•   •  •••  "«• 


••    ••.*•!  -  ."   *    .  • 


5tanbop€  iprcsa 

H.   CILSON     COMPANY 
BOSTON.     U.S.A. 


PEEFACE 


THE  reader  of  this  book  will  understand  from  its  contents,  and 
particularly  from  its  concluding  chapter,  why  it  has  remained  for 
one  of  Borders  pupils  to  present  his  work  as  a  whole  to  the  scientific 
world.  Although  for  more  than  fifteen  years  a  protagonist  in  the 
modern  development  of  Immunity,  Bordet  has  continued  an  in- 
vestigator instead  of  becoming  a  generalizer;  he  has  been  led  by 
thoroughness  of  observation  and  brilliancy  of  inductive  reason  to 
the  collection  of  successive  significant  facts  rather  than  to  the 
assimilation  of  scattered  data  in  support  of  a  preconceived  theory. 
Whereas  others  have  been  willing  to  venture  more  fully  explanatory 
theories  of  Immunity,  Bordet,  although  contributing  a  dispropor- 
tionate number  of  important  facts,  has  contented  himself  with  the 
hypotheses  which  bridge  from  one  experiment  to  another,  and  has 
fully  realized  the  necessarily  fragmentary  nature  of  our  present 
knowledge  in  this  field  of  science. 

This  compilation  was  undertaken,  not  so  much  owing  to  the 
apparent  demand  for  similar  examples  of  monographic  collection, 
as  from  a  sincere  conviction  of  the  fundamental  importance  of  each 
contribution  which  Jules  Bordet  has  directly  or  indirectly  given  us. 
The  somewhat  extensive  task  has  been  welcomed  as  a  means  of 
expressing  gratitude  for  a  personal  inspiration  in  scientific  thought 
and  method. 

It  would  seem  of  particular  importance,  in  view  of  the  over- 
Germanizing  of  American  science,  that  this  collection  of  monographs 
should  appear  in  this  country. 

The  volume  includes  all  of  Bordet's  scientific  contributions  with 
a  few  exceptions.  Among  the  latter  may  be  mentioned  the  article 
with  Danysz  on  the  method  of  combined  active  and  passive  im- 
munization against  Rinderpest,  a  series  of  four  articles  on  Coagula- 
tion of  Blood  (with  Gengou),  and  a  note  on  the  Spirocheta  pallida, 

26^735 


iv  PREFACE. 

which  organism  Bordet  and  Gengou  were  incidentally,  the  first  to 
see.  The  articles  have  in  general  been  arranged  in  chronological 
order;  an  exception,  however,  was  made  in  some  of  the  latter 
chapters  (XXIV  to  XXX)  which  deal  rather  with  the  application 
of  more  theoretical  studies  which  precede. 

I  am  indebted  to  Professor  Bordet,  not  only  for  consenting  to  the 
collection  of  these  articles,  but  also  for  furnishing  the  concluding 
chapter  and  for  certain  suggestions  and  corrections.  Doctor 
Gengou  was  so  kind  as  to  abstract  one  of  his  articles  for  this  publi- 
cation. Thanks  are  also  due  to  the  publishers  of  the  various  jour- 
nals in  which  these  articles  appeared  for  permission  to  translate  or 
to  reprint  them. 

F.  P.  G. 

BOSTON,  August,  1909 


TABLE  OF  CONTENTS. 


(From  the  Laboratory  of  Professor  Errera,   Botanical  Institute, 

Brussels.} 

CHAPTER  PAGE 

I.   The  Adaptive  Changes  of  Bacterial  Cultures  in  the  Body  of 

Immunized  Animals 1 

BORDET. 

(From  the  Laboratory  of  Professor  Metchnikoff,  Pasteur  Institute, 

Paris.) 
II.   Studies  on  the  Serum  of  Vaccinated  Animals.     The  Relation  of 

Leucocytes  to  Immunity 8 

BORDET. 

III.  Studies    on    the    Serum    of    Vaccinated    Animals.     Pfeiffer's 

phenomenon 56 

BORDET. 

IV.  On  the  Mode  of  Action  of  Preventive  Sera 81 

BORDET. 

V.   A  Contribution  to  the  Study  of  Antistreptococcus  Serum 104 

BORDET. 

VI.    On  the  Agglutination  and  Dissolution  of  Red  Blood  Cells  by  the 

Serum  of  Animals  Injected  with  Defibrinated  Blood 134 

BORDET. 

VII.   The  Mechanism  of  Agglutination 142 

BORDET. 

VIII.   The  Agglutination  and  Dissolution  of  Red  Blood  Cells  by  Serum 

(Second  Memoir) 165 

BORDET. 

IX.   Hemolytic  Sera  and  Their  Antitoxins;  and  Theories  Concerning 

Cytolytic  Sera  in  General 186 

BORDET. 
v 


I  TABLE  OF  CONTENTS. 

CHAPTER  ,  PAGE 

X.   On  the  Existence  of  Sensitizing  Substances  in  the  Majority  of 

Antimicrobial  Sera 217 

BORDET   AND    GENGOU. 

XI.   On  the  Mode  of  Action  of  Cytolytic  Sera;  and  on  the  Unity  of  the 

Alexin  in  a  Given  Serum 228 

BORDET. 

(From  the  Pasteur  Institute  of  Brabant,  Brussels.) 

XII.   On  the  Sensitizers  of  Sera  Active  against  Albuminous  Substances    241 

GENGOU. 

XIII.  On  the  Mode  of  Action  of  Antitoxins  on  Toxins 259 

BORDET. 

XIV.  The  Properties  of  Antisensitizers  and  the  Chemical  Theories  of 

Immunity 280 

BORDET. 

XV.   Researches  on  the  Agglutination  of  Red  Blood  Cells  by  Chemical 
Precipitates,  and  on  the  Suspension  of  Such  Precipitates  in 

Colloidal  Media , 312 

GENGOU. 

XVI.    Observations  on  the  Single  Nature  of  Hemolytic  Immune  Bodies 

and  on  the  Existence  of  So-called  "  Complementoids  " 333 

GAY. 

XVII.  The  Fixation  of  Alexins  by  Specific  Serum  Precipitates 346 

GAY. 

XVIII.   Deviation  of  the  Alexin  in  Hemolysis 357 

GAY. 


IX.   On  the  Relations  of  Sensitizers  to  Alexin 363 

BORDET  AND  GAY. 

XX.   On  the  Nature  of  Opsonins 389 

SLEESWIJK. 

XXI.   Alexin  Absorption  and  the  Antagonistic  Property  of  Normal  Sera     398 
BORDET  AND  GAY. 

XXII.   A  Contribution  to  the  Study  of  Molecular  Adhesion,  with  a  con- 
sideration of  its  Function  in  Various  Biological  Phenomena.  .      Ill 
GENGOU. 


TABLE   OF  CONTENTS.  vii 

CHAPTER  PAGE 

XXIII.  The  Phenomena  of  Adsorption  and  the  Conglutinin  of  Bovine 

Serum 440 

BORDET    AND    STRENG. 

XXIV.  Sensitizers  for  the  Tubercle  Bacillus 462 

BORDET  AND  GENGOU. 

XXV.    New  Contribution  to  the  Study  of  Sensitizers  for  Tubercle  Bacilli     464 

GENGOU. 

XXVI.    A  Contribution  to  Our  Knowledge  of  Antituberculous  Sensitizers     467 

GENGOU. 

XXVII.   The  Bacillus  of  Whooping  Cough 472 

BORDET  AND  GENGOU. 

XXVIII.   An  Additional  Note  on  the  Whooping-Cough  Bacillus 482 

BORDET  AND  GENGOU. 

XXIX.   The  Endotoxin  of  the  Whooping-Cough  Bacillus 488 

BORDET. 

XXX.   Researches  on  Avian  Diphtheria 492 

BORDET. 

XXXI.    A  General  R<§sum<§  of  Immunity 496 

BORDET. 

Index  of  Authorities  quoted 531 

Index  of  Subjects 535 


STUDIES  IN  IMMUNITY. 


I.  THE  ADAPTIVE  CHANGES  OF  BACTERIAL  CULTURES 
IN  THE  BODY  OF  IMMUNIZED  ANIMALS  * 

BY  JULES  BORDET,  STUDENT  OF  MEDICINE. 

BACTERIA  are  highly  adaptable.  They  frequently  change  both 
morphologically  and  functionally.  Their  virulence  is  also  an  essen- 
tially fluctuating  property,  that  increases  or  diminishes  according 
to  the  conditions  to  which  the  pathogenic  organism  is  subjected. 

The  vibrio  Metchnikovi,  as  described  by  Gamaleia,  is  very  virulent 
for  certain  animals.  A  small  amount  of  culture  suffices  to  kill 
guinea-pigs.  Sterilized  bouillon  in  which  the  organism  has  been 
grown  is  also  very  toxic  for  these  animals  and  kills  in  a  mean  dose 
of  2  c.c.  to  100  grams  of  body  weight. 

Guinea-pigs,  although  they  succumb  to  this  micro-organism  so 
easily,  become  immunized  when  they  have  received  one  or  two 
injections  of  sufficient  quantities  0.5  to  1  c.c.  per  100  grams)  of  a 
culture  freed  from  living  bacteria  by  filtration  or  by  autoclaving. 
The  protection  obtained  in  this  manner  is  constant. 

Metchnikoff  has  shown  us,  however,  that,  when  a  guinea-pig  pro- 
tected in  this  manner  is  given  a  small  amount  of  a  virulent  culture 
of  living  organisms,  the  latter  are  not  immediately  destroyed. 
They  disappear  only  after  a  relatively  long  period,  60  to  90  hours 
if  injected  subcutaneously,  or  6  to  7  days  when  injected  into  the 
anterior  chamber  of  the  eye.  There  is,  however,  a  much  more 
notable  collection  of  leucocytes  at  the  point  of  inoculation  than  in 
an  animal  that  has  not  been  protected  against  the  disease.  Not 
only  do  the  vibrios  remain  alive,  but  they  lose  none  of  their  harmful 

.*  Adaptation  des  virus  aux  organismes  vaccines.  Annales  de  Tlnstitut  Pas- 
teur, VI,  1892,  28.  From  the  laboratory  of  Prof.  Errera.  Botanical  Institute, 
Brussels. 

1 


L>  STUDIES  IN  IMMUNITY. 

properties;  on  the  contrary,  their  virulence  is  to  a  considerable 
extent  increased.  With  an  organism  modified  in  this  manner, 
Metchnikoff  has  succeeded  in  killing  a  guinea-pig  in  from  G  to  7 
hours,  whereas  the  ordinary  culture  takes  about  20  hours. 

What  is  the  cause  of  this  marked  increase  in  pathogenicity?  By 
what  modification  has  the  virus  acquired  this  powerful  activity? 
This  point  we  have  attempted  to  determine. 

Let  us  immunize  a  guinea-pig  of  550  grams  weight  by  injecting 
3.5  c.c.  of  a  sterilized  culture  of  V.  Metchnikovi,  followed  8  clays 
later  by  0.5  c.c.  of  a  living  culture  of  the  organism.  Ten  days  later, 
when  the  animal  has  returned  to  a  normal  condition,  we  introduce 
1  c.c.  of  a  vigorous  culture  under  the  skin  of  the  belly.  Twenty 
hours  later  a  small  amount  of  the  exudate  which  has  been  formed 
is  withdrawn  and  grown  in  veal  bouillon  containing  peptone. 

When  this  culture  has  grown  out,  a  cubic  centimeter  of  it  is  taken 
and  injected  subcutaneously  in  another  similarly  vaccinated  guinea- 
pig.  Twenty-seven  hours  after  inoculation,  a  drop  of  the  exudate 
is  withdrawn  as  from  the  first  animal  and  likewise  grown  in  bouillon. 

This  second  guinea-pig,  although  previously  in  good  condition,  is 
much  sicker  than  the  first  vaccinated  animal.  There  is  a  large  area 
of  necrosis  about  the  point  of  inoculation;  the  eyes  are  half  closed, 
the  animal  inert,  and  prostration  manifest.  This  is  in  harmony 
with  the  experiments  of  Metchnikoff,  who  demonstrated  that  the 
vibrio  that  had  remained  under  the  skin  of  a  vaccinated  guinea-pig 
for  20  hours  became  more  pathogenic. 

We  have  now  three  different  cultures  of  the  vibrio  Metchnikovi :  a 
culture  of  the  organism  modified  by  a  single  passage  through  an 
immunized  animal;  a  culture  of  the  same  organism  modified  by  two 
passages;  and  a  culture  of  the  vibrio  grown  on  agar  in  successive 
generations  during  several  months.  We  may  obtain  a  fourth  cul- 
ture by  passing  the  vibrio  through  a  non-vaccinated  guinea-pig. 
This  last  constitutes  a  culture  of  normal  vibrios  whose  virulence  has 
undergone  no  modification  through  prolonged  growth  on  artificial 
media. 

The  increase  in  virulence  of  cultures  which  we  call  "  modified," 
or  very  virulent,  may  be  due,  according  to  our  present  ideas,  to  one 
of  two  causes:  either  their  chemiotactic  power  toward  leucocytes 
has  diminished,  which  would  allow  them  to  escape  more  readily 


ADAPTIVE  CHANGES  OF  BACTERIAL  CULTURES.  3 

the  destructive  action  of  these  cells  and  consequently  develop  and 
multiply  with  greater  security;  or,  indeed,  they  may  secrete  more 
abundant  or  more  dangerous  toxic  products  than  before. 

These  two  causes  may  operate  simultaneously.  It  is  necessary 
then  to  compare,  first,  the  chemiotactic  influence  of  the  original  or 
normal  organism  with  that  of  the  "modified"  organism,  and  second, 
the  toxic  properties  of  the  substances  formed  by  these  two  strains 
of  vibrios. 

EXPERIMENT  1.  The  -four  different  organisms  were  grown  in  a 
special  fluid  that  offers  a  good  culture  medium  which  has  already 
been  used  by  J.  Massart  and  Ch.  Borclet;  this  medium  has  in  itself 
not  the  slightest  chemiotactic  effect  on  leucocytes. 

After  three  days  of  growth  at  32°  C.,  capillary  tubes  are  filled 
with  these  virulent  cultures  and  placed  in  lots  of  a  dozen  in  the 
peritoneal  cavity  of  a  normal  guinea-pig.  They  are  removed  8 
hours  later,  when  it  is  found  that  the  columns  of  leucocytes  that 
have  entered  the  tubes  vary  in  length.  In  the  tube  containing  the 
normal  vibrio,  cultivated  for  several  months  on  agar,  the  length  of 
this  column  is  the  same  as  with  the  normal  vibrio  recently  isolated 
from  the  tissues  of  an  unvaccinated  guinea-pig.  With  the  vibrio 
modified  by  a  single  passage  through  a  vaccinated  guinea-pig  the 
column  is  about  half  that  of  the  preceding.  With  the  organism 
modified  by  two  passages  it  is  slightly  less  than  half. 

The  same  experiment  was  performed  with  cultures  sterilized  at 
115°  C.  No  difference  in  the  influx  of  leucocytes  was  noted. 

It  is  evident,  then,  that  the  attracting  power  for  leucocytes  is 
notably  less  in  organisms  grown  in  immunized  guinea-pigs  than  in 
the  two  normal  organisms;  one  that  was  passed  through  a  normal 
guinea-pig  and  the  other  grown  for  a  long  time  on  artificial  media. 

It  is  rather  interesting  to  note  that  tnere  is  no  appreciable  dif- 
ference between  these  last  two  strains  of  vibrio.  The  chemiotactic 
properties  evidently  have  not  been  altered  by  growing  on  agar. 

What  is  more,  it  may  be  mentioned  that  we  have  found  that  the 
vibrio  Metchnikovi  in  old  cultures  exposed  to  the  air  is  little,  if  at  all 
attenuated;  a  culture  bearing  the  date  Dec.  16, 1891,  and  inoculated 
March  29,  1892,  in  a  dose  of  0.5  c.c.,  killed  guinea-pigs  in  20  hours. 
As  is  well  known,  many  pathogenic  organisms  attenuate  more 
rapidly. 


4  STUDIES  IN  IMMUNITY. 

These  experiments  show  us,  moreover,  that  a  vibrio  that  has 
undergone  two  passages  is  only  slightly  less  attracting  than  one 
that  has  undergone  a  single  passage. 

Let  us  now  consider  whether  the  toxicity  of  the  bacterial  secre- 
tions has  changed  in  a  manner  similar  to  that  of  the  chemiotactic 
property. 

EXPERIMENT  2.  A  healthy  guinea-pig,  weighing  620  grams,  was 
given  an  inoculation  of  6  c.c.  of  a  very  virulent,  modified,  sterilized 
culture.  Although  this  amount  was  rather  small,  it  killed  the 
animal  in  60  hours.  At  autopsy  no  lesions  other  than  intestinal 
congestion  were  noted. 

Another  guinea-pig,  weighing  450  grams,  received  5.5  c.c.  of  the 
ordinary  vibrio  Metchnikovi,  which  in  proportion  to  the  weight  of 
the  animal  was  slightly  more  than  was  given  the  preceding  animal. 
This  animal  showed  no  effect,  however,  and  remained  in  good  condi- 
tion. 

It  is  to  be  noted  that  an  increase  of  toxicity  here  accompanies  a 
lowering  in  positive  chemiotactic  power. 

To  what  is  this  weakness  in  attraction  due?  Are  we  to  regard  it 
simply  as  a  quantitative  decrease  in  the  attracting  substance  that 
the  normal  vibrio  produces  so  abundantly?  Or  is  there  formed,  on 
the  contrary,  a  repelling  substance  of  which  little  or  none  is  formed 
by  the  original  vibrio?  An  attempt  to  reply  to  these  questions 
follows. 

EXPERIMENT  3.  We  placed  in  the  peritoneal  cavity  of  a  normal 
guinea-pig  capillary  tubes  containing  the  following  vibrios,  pre- 
pared on  the  same  culture  media  as  in  experiment  1 : 

(a)  Normal  sterilized  culture  of  vibrio  Metchnikovi.  (b)  Steril- 
ized culture  of  the  vibrio  Metchnikovi  from  the  exudate  withdrawn 
from  the  second  immunized  guinea-pig,  (c)  A  mixture  of  equal 
parts  of  the  two  first  fluids,  (d)  A  mixture  of  equal  parts  of  fluid 
"b"  and  sterile  culture  medium,  identical  with  that  on  which 
the  organisms  had  been  grown. 

These  tubes  were  left  in  the  animal  body  for  8  hours.  On  exami- 
nation the  columns  of  leucocytes  in  tubes  containing  liquids  "a," 
"c,"  and  "d"  were  seen  to  be  equal.  In  the  "b"  tubes  the  length 
of  the  column  of  leucocytes  was  less  than  half  this. 

A  consideration  of  tube  "d"  shows  us  that  dilution  with  an  inert 


ADAPTIVE  CHANGES  OF  BACTERIAL  CULTURES.  5 

fluid  increases  the  attracting  power  of  the  " modified"  vibrio  Metch- 
nikovi  and  renders  it  quite  as  energetic  as  the  normal  organism. 

We  may  then  put  aside  the  hypothesis  that  the  modified  vibrio 
has  lost  a  part  of  its  attracting  properties  owing  to  a  smaller  amount 
of  positive  chemiotactic  substance.  There  is  evidently  a  negative 
chemiotactic  influence  which  lessens  to  a  considerable  degree  the 
effect  of  the  attractive  substances.  Experiment  shows  us  that 
dilution  weakens  the  activity  of  the  first  more  than  that  of  the 
second. 

There  is  nothing  to  prove  that  these  substances,  acting  in  an 
opposite  manner  on  white  corpuscles,  are  distinct.  It  may  be  that 
there  are  not  two  different  chemical  substances,  but  that  one  and 
the  same  substance  may  attract  leucocytes  when  diluted  and  repel 
them  when  concentrated.  This  last  hypothesis  is  far  from  difficult 
to  accept:  we  know,  indeed,  the  profound  influence  which  the  con- 
centration of  various  substances  has  on  their  power  to  affect  sus- 
ceptible cells.  This  is  not  a  supposition,  however,  that  we  have  been 
able  to  prove. 

Whatever  the  substance  may  be  to  which  the  repelling  action  is 
due,  it  is  present  and  acts  energetically  in  the  modified  vibrio,  but 
it  is  also  manifest  to  a  less  degree  in  the  ordinary  organism.  As  a 
matter  of  fact,  the  attracting  power  of  the  secretions  of  this  latter 
organism  increases  with  dilution,  as  demonstrated  by  a  capillary 
tube  experiment. 

The  vibrio  that  has  been  grown  in  an  immunized  guinea-pig  does 
not  long  retain  its  intense  repelling  property  when  grown  on  arti- 
ficial media. 

EXPERIMENT  4.  A  comparison  was  made  as  regards  chemio- 
taxis,  by  the  same  method  of  capillary  tubes,  between  the  vibrio 
Metchnikovi  cultivated  for  a  long  time  on  agar  tubes  and  a  vibrio 
marked  "very  virulent"  that  had  been  transplanted  every  four 
days  for  nearly  half  a  month  in  veal  bouillon  containing  1  per  cent 
peptone.  The  leucocytes  filled  equal  lengths  of  the  tubes  of  each 
strain. 

We  have  seen  that  residence  in  the  body  of  a  vaccinated  animal 
lends  certain  modifications  to  the  vibrio  Metchnikovi;  it  becomes 
more  toxic  and  less  attracting  for  leucocytes.  Let  us  now  consider 
how  these  modifications  are  produced. 


6  STUDIES  IN   IMMUNITY. 

When  we  inject  vibrios  subcutaneously  in  a  vaccinated  guinea- 
pig  an  emigration  of  leucocytes  rapidly  takes  place.  The  first  white 
corpuscles  to  arrive  at  the  point  of  inoculation  find  a  considerable 
number  of  adversaries  of  which  they  can  destroy  only  a  few.  It 
would  be  sufficient  for  a  few  of  these  vibrios  to  be  endowed  with  a 
slightly  more  intense  power  of  attraction  than  their  fellows,  to  cause 
the  phagocytes  to  direct  themselves  by  preference  toward  these 
organisms  and  to  take  them  up.  Very  slight,  almost  inappreciable 
differences  will  consequently  predestine  certain  bacteria  to  rapid 
phagocytosis.  In  the  same  way  an  inferiority  in  secretion  of  toxic 
products,  however  slight,  will  cause  a  predisposition  to  rapid 
destruction. 

In  a  word,  leucocytes  kill  first  those  organisms  that  are  less  resist- 
ant, and  the  culture  inoculated  will  be  freed  first  of  those  individuals 
which  either  form  less  poison  than  their  fellows  or  attract  the 
leucocytes  more. 

In  the  meantime  the  vibrios  that  have  been  left  alone  will  divide 
and  produce  new  organisms  which  in  turn  will  be  exposed  to  the 
attack  of  phagocytes.  These  latter  will  again  suppress  those  indi- 
viduals that  are  most  poorly  armed  for  the  struggle,  and  will  leave 
only  those  that  possess  in  highest  degree  the  two  characters  just 
noted.  Thanks  to  this  process  of  selection,  new  generations  of 
organisms,  like  those  represented  by  the  cultures  used  in  our  experi- 
ments, will  be  derived  for  the  most  part  from  those  bacteria  which 
have  been  endowed  with  certain  advantages. 

For  the  production  of  this  new  race  it  is  necessary: 

First :  that  leucocytes  come  up  continually.  If  they  do  not  come 
to  the  point  of  inoculation,  no  selection  occurs  and  the  organisms  do 
not  gain  in  pathogenicity.  So,  when  virulent  vibrios  are  injected 
subcutaneously  in  a  normal  guinea-pig,  the  influx  of  leucocytes  is 
very  slight  and  the  organism  obtained  by  this  passage  has  no  par- 
ticular characteristics.  If,  on  the  contrary,  a  normal  guinea-pig  is 
given  an  inoculation  of  an  attenuated  vibrio  Metchnikovi,  which  has 
a  strong  attraction  for  leucocytes,  the  selection  is  brought  about,  and 
the  organism  increases  in  virulence.  The  leucocytes  are  the  active 
agents  in  selection,  and  it  is  they  that,  by  eliminating  the  least 
resistant  organisms  and  sparing  the  others  for  a  while  so  that  they 
may  multiply,  cause  the  increase  in  virulence  that  has  been  noted. 


ADAPTIVE  CHANGES  OF  BACTERIAL  CULTURES.       7 

Second :  that  the  influx  of  leucocytes  take  place  with  a  certain 
regularity  and  in  a  gradual  and  progressive  manner.  Selection 
requires  a  certain  time.  It  is  in  those  places  in  an  immunized 
animal  where  the  influx  of  leucocytes  occurs  slowly,  for  example, 
in  the  anterior  chamber  of  the  eye,  that  the  vibrio  Metchnikovi 
acquires  the  most  extreme  virulence.  It  has  been  noted  in  referring 
to  Metchnikoff's  work  that  the  vibrio  obtained  in  this  manner  is  the 
most  active,  killing  the  guinea-pig  in  6  hours. 

Another  fact  of  Metchnikoff's  is  very  similar.  Anthrax  is  more 
deadly  for  the  pigeon  when  injected  into  the  aqueous  humor  than 
elsewhere.  It  will  be  recalled  also  that  with  some  organisms  inocu- 
lation into  the  circulation  is  much  less  dangerous  than  into  the 
connective  tissue. 

This  ends  our  account  of  the  vibrio  Metchnikovi  for  the  present; 
we  shall  later  return  to  it: 

Not  all  organisms,  as,  for  example,  the  bacillus  of  swine  plague 
(rouget  du  pore),  acquire  the  same  virulence  when  passed  through 
vaccinated  or  more  or  less  refractory  animals. 

New  studies  to  determine  whether  a  culture  subjected  to  various 
conditions  becomes  modified  in  respect  to  its  chemiotactic  power 
and  the  toxicity  of  its  secretions  are  in  progress. 


II.  STUDIES  ON  THE  SERUM  OF  VACCINATED 
ANIMALS* 

BY   DR.   JULES   BORDET. 

The  subject  of  immunity  has  expanded  in  the  last  few  years. 
The  fundamental  problem,  to  the  solution  of  which  a  considerable 
number  of  researches  have  been  devoted,  is  to  explain  how  animals, 
either  on  account  of  a  natural  refractory  condition  or  owing  to  an 
artificial  immunization,  resist  the  invasion  of  a  given  micro-organism 
and  overcome  an  infection  by  destroying  the  bacterium  or  the 
poison  which  it  secretes.  But  there  are  other  facts  which  also 
demand  explanation.  Animals  which  have  been  well  immunized 
against  a  given  infection  either  by  repeated  injections  of  bacterial 
products  or  of  living  cultures  may  be  distinguished  from  normal 
animals  not  only  by  the  fact  that  they  henceforth  resist  this  infec- 
tion, but  also,  in  numerous  instances,  by  the  fact  that  their  body 
fluids,  particularly  the  blood  serum,  possess  properties  which  are 
not  observed  in  the  non-vaccinated  animal.  The  serum  of  vacci- 
nated animals  is  often  preventive  and  at  times  bactericidal  or 
antitoxic.  It  is  not  sufficient  for  theories  of  immunity  to  explain 
how  an  animal  that  has  been  vaccinated  is  fitted  to  overcome 
an  infection;  they  must  also  explain  how  these  new  properties, 
the  study  of  which  has  so  greatly  interested  bacteriologists, 
have  been  formed  in  the  body  fluids.  The  study  of  the  serum  of 
immunized  animals  forms  a  new  chapter  in  the  history  of  the 
struggle  between  the  animal  and  infective  agents,  under  which  head- 
ing practical  results  of  the  highest  importance  are  already  inscribed. 
Any  explanation  of  the  phenomena  is,  however,  still  far  from 
complete. 

The  substances  which  are  present  in  the  serum  of  vaccinated 
animals  and  which  endow  them  with  their  particular  property  are 

*  Contribution  &  lY'tude  du  serum  chez  les  animaux  vaccines.  Annales  de  la 
Societ^  royale  des  sciences  me*dicales  et  nat.  de  Bruxelles,  IV,  1895. 

8 


STUDIES  ON  THE  SERUM  OF   VACCINATED  ANIMALS.          9 

unknown  to  us.  But  apart  from  their  chemical  constitution,  con- 
cerning which  hypotheses  only  may  be  offered,  these  substances 
should  be  studied  from  various  points  of  view,  and,  indeed,  innumer- 
able questions  concerning  their  functions  and  their  origin  at  once 
suggest  themselves.  Where  are  these  materials  formed?  By  what 
cells  are  they  elaborated?  Are  they  uniformly  distributed  and 
dissolved  in  the  body  fluids;  or  are  they  concentrated  in  certain 
individual  regions?  What  is  their  function  in  acquired  immunity? 
To  what  extent  does  bactericidal  power  contribute  to  protect  the 
organism?  By  what  mechanism  do  preventive  substances  in  sera 
insure  immunity  in  animals  in  which  they  have  been  injected? 
Certain  of  these  problems  have  already  occupied  the  attention  of 
experimenters;  certain  of  them  have  been  attacked  with  more  or 
less  success  and  treated  more  or  less  completely,  and  others  have 
only  been  touched  on.  Without  any  premature  hope  of  ultimate 
explanation,  one  may  extend  our  knowledge  by  offering  new  facts, 
or  contributing  a  detail. 

LEUCOCYTES  AND  IMMUNITY. 

I.   PHAGOCYTOSIS  IN  GENERAL.     PHAGOCYTOSIS  IN  CHOLERA. 

The  studies  which  are  to  be  detailed  in  the  present  article  lead  us 
to  attribute  to  the  leucocytes  an  important  function  in  the  elabora- 
tion of  those  substances  which  endow  sera  with  their  activity.  It 
has  been  known  for  some  time,  thanks  to  the  persevering  efforts  of 
the  creator  of  the  existing  theory  of  immunity,  Metchnikoff,  that 
body  cells  take  an  energetic  part  in  dissipating  an  infection  and  so 
aid  in  a  very  important  manner  in  the  cure  of  infectious  diseases. 
Leucocytes  respond  to  stimuli  in  several  ways  and  are  capable  of 
reacting  in  several  different  manners.  These  different  reactions 
are  often  all  exercised  in  the  course  of  a  given  phenomenon.  They 
are  often  all  necessary  for  the  accomplishment  of  a  single  vital  act. 
The  study  of  these  properties  forms,  then,  a  collective  whole  which 
may  not  be  dissociated,  and  for  this  reason  it  seems  useful  to  review 
at  the  beginning  whatever  is  known  concerning  the  participation 
of  the  white  blood  corpuscles  in  the  struggle  against  bacterial 
parasites. 

The  fundamental  facts  of  the  phagocytic  theory  have  been  so 


10  STUDIES  IN   IMMUNITY. 

frequently  determined  by  individual  researches,  so  frequently 
demonstrated  in  articles  dealing  with  the  general  problem  of 
immunity,  that  it  would  scarcely  be  necessary  to  repeat  the  history 
of  its  development  and  to  enumerate  the  data  on  which  it  has  been 
founded.  We  may  be  permitted,  however,  to  recall  the  most 
demonstrative  facts  which  may  be  cited  in  confirmation  of  this 
doctrine. 

The  phagocytic  function  belongs  essentially  to  the  polynuclear 
amphophiles,*  to  the  large  mononuclear  leucocytes  and  to  certain 
endothelial  cells  of  the  capillaries  of  the  liver  and  the  spleen  (their 
parenchymatous  cells  may  also  in  certain  cases  manifest  phagocytic 
properties).  Phagocytosis  is  found  throughout  the  animal  king- 
dom. It  occurs  in  all  the  contagious  diseases  that  have  been  well 
studied,  not  only  in  those  of  a  particularly  infectious  nature,  but 
also  in  those  that  are  preeminently  toxic  and  in  which  bacterial 
invasion  is  extremely  limited.  The  tetanus  bacillus  and  the 
diphtheria  bacillus,  for  example,  are  taken  up  and  destroyed  by 
phagocytes.  Phagocytes  also  play  an  essential  role  in  chronic 
bacterial  infections,  as  the  constitution  of  the  tubercle  indicates. 

Phagocytes  do  not  limit  their  activities  to  the  taking  up  of  living 
or  dead  microbes.  They  may  also  capture  spores  and  prevent  their 
germination.  Their  activity  may  also  be  utilized  against  other 
foreign  bodies  and  against  the  other  cells  of  the  same  animal  when 
they  have  become  useless  or  even  harmful  and  are  destined  to 
disappear.  The  protoplasm  of  phagocytes  likewise  takes  up  soluble 
substances  introduced  into  the  body,  as  was  shown  by  the  researches 
of  Samoiloff,t  and  of  Lipski,J  (Pharmacological  Institute  of  Dorpat) 
on  the  distribution  of  iron  and  of  silver  as  soluble  compounds  in  the 
animal  body. 

What  proves  the  very  fundamental  function  of  phagocytes  in  the 
destruction  of  bacteria  is  the  surprising  rapidity  with  which  the 
engulfing  of  organisms  may  be  accomplished,  as  several  observers, 

*  Eosinophilic  leucocytes  only  rarely  take  up  bacteria.  Mesnil  (Annales  de 
1'Institut  Pasteur,  May,  1895)  has  noted,  in  the  frog,  eosinophilic  cells  in  the  act 
of  engulfing  anthrax  bacilli.  We  have  still  more  recently  noted  the  taking  up 
of  the  diphtheria  bacillus  by  the  eosinophiles  of  a  guinea-pig  immunized  by  a 
strong  dose  of  anti-diphtheria  serum. 

t  Samoiloff,  Arbeiten  des  pharmakologischen  Institutes  zu  Dorpat,  IX,  1893. 

t  Lipski,  Ibid. 


STUDIES  ON  THE  SERUM   OF   VACCINATED  ANIMALS.         11 

and  Werigo  *  in  particular,  have  shown.  We  shall  have  occasion  to 
consider  the  taking  up  by  phagocytes  of  cholera  vibrios  injected 
intravenously.  We  have  already  noted  the  almost  instantaneous 
occurrence  of  this  phenomenon. 

The  intensity  with  which  phagocytosis  occurs  in  infected  animals 
is  always  proportionate  to  the  resistance  of  the  animal;  in  the 
majority  of  cases  it  may  serve  in  the  course  of  an  infection  as  an 
indication  of  the  outcome  of  the  struggle  between  the  bacterium 
and  the  animal.  So,  for  instance,  it  is  more  active  in  vaccinated 
animals  than  in  normal  animals,  whether  the  refractory  stage  be 
obtained  as  a  result  of  the  injection  of  attenuated  or  sterilized 
cultures  or  by  means  of  a  preventive  serum. 

The  existence  of  a  peculiar  reaction  on  the  part  of  leucocytes  to 
chemical  substances  secreted  by  bacteria  offers  important  evidence 
of  the  part  which  these  cells  take  in  defense  of  the  body.|  As  soon 
as  the  bacteria  penetrate  the  tissue  their  presence  is  evident  from 
the  fact  that  their  diffusible  products  cause  a  phenomenon  of  chemi- 
otaxis  on  the  part  of  the  phagocytes.  The  tactile  reaction  of  these 
cells  permits  them,  when  chemiotaxis  has  been  manifested  and  when 
they  have  approached  the  infecting  bacteria  to  the  point  of  contact, 
to  send  elongations  about  them  and  to  engulf  them.  The  rapidity 
with  which  these  various  phenomena  follow  each  other,  particu- 
larly in  vaccinated  animals  or  when  dealing  with  slightly  virulent 
organisms,  shows  how  active  phagocytic  intervention  is. 

The  relation  between  the  quality,  method  of  reaction,  and  number 
of  phagocytes  on  the  one  hand  and  the  efficiency  of  the  defense  on 
the  other  is  shown  by  a  series  of  phenomena.  Leucocytes  are  little 
attracted,  and  may  even  be  repulsed  by  very  virulent  microbes. 

An  attempt  has  recently  been  made  to  discredit  the  importance 
of  chemiotaxis  in  immunity.  Werigo,  who  of  course  recognizes 
the  existence  and  the  purpose  of  positive  chemiotaxis,  has  certain 
doubts  as  to  the  existence  of  a  negative  chemiotaxis.  Woronin 
goes  even  further.  He  attributes  to  chemiotaxis,  whether  positive 
or  negative,  an  almost  negligible  function  in  the  defense  of  the 

*  Werigo,  Les  globules  blancs  protecteurs  du  sang:  Annales  de  Tlnstitut 
Pasteur,  1892. 

t  J.  Massart  et  Ch.  Bordet,  Journal  de  la  Societe  royale  des  sciences  naturelles 
et  medicales  de  Bruxelles,  1890. 


12  STUDIES  IN   IMMUNITY. 

organism  against  infection.  It  is  certain  that  positive  chemiotaxis 
of  leucocytes  has  been  proved  in  an  exact  manner  by  demonstrative 
experiments  that  cannot  be  controverted.  Bacterial  cultures 
attract  leucocytes,  and  this  attraction  is  often  very  marked.  This 
is  an  established  fact,  the  significance  of  which  from  the  standpoint 
of  immunity  appears  quite  evident.  Let  us  hasten  to  add  that 
Woronin  does  not  deny  chemiotaxis;  but  he  thinks  that  this  re- 
action on  the  part  of  the  leucocytes  is  not  indispensable  nor  even 
particularly  useful  to  them.  Phagocytes  may,  according  to  this 
author,  fulfil  their  function  simply  by  means  of  their  tactile  reaction. 
This  investigator  attempts  to  show  anew  the  already  well  known 
fact  that  leucocytes  do  react  to  contact,  that  they  voluntarily  enter 
porous  bodies,  openings  in  tissues,  etc.  He  recognizes  that  the 
reaction  to  contact  is  very  highly  developed  and  concludes  that  the 
engulfing  of  bacteria  may  be  due  simply  to  this  reaction.  No  one 
doubts  that  the  tactile  reaction  is  a  valuable  property  in  leucocytes, 
as  is  made  clear  by  those  authors  who  have  studied  chemiotaxis.* 
It  appears  quite  certain  that  phagocytes,  when  they  open  up  on 
contact  with  inert  non-attractive  particles  (charcoal,  for  example), 
may  ingest  them  in  their  protoplasm ;  this  ingestion  is  due  entirely 
to  the  tactile  sensitiveness  of  the  cells.  But  how  does  the  import- 
ance of  the  leucocytic  reaction  to  contact  diminish  the  reaction  to 
chemical  substances?  How  much  must  the  taking-up  of  living  or 
dead  particles  be  facilitated  if  these  particles  diffuse  substances 
that  attract  leucocytes  and  cause  them  to  move  toward  the  particles? 
If  we  admit  the  intensity  of  positive  chemiotaxis,  which  is  easily 
determined,  we  cannot  deny  to  these  phenomena  their  evident 
function.  Werigo 's  objections  f  to  the  arguments  which  tend  to 
admit  a  negative  chemiotaxis  of  leucocytes  are  better  founded. 
Werigo  does  not  think  it  has  been  shown  that  there  are  substances 
capable  of  producing  a  retraction  of  leucocytes.  We  have  been 
accustomed  to  regard  lactic  acid,  for  example,  as  possessing  such  a 
repellent  action  from  the  following  experiment:  if  we  mix  a  small 
quantity  of  lactic  acid  with  substances  which  are  very  attractive 
(bacterial  products),  it  is  noted  that  the  mixture  no  longer  attracts 

*  J.  Massart  and  C.  Bordet.,  loc.  cit. 

f  Werigo,  De"veloppement  du  charbon  chez  le  lapin.     Annales  de  1'Institut 
Pasteur,  1894. 


STUDIES  ON  THE  SERUM   OF  VACCINATED  ANIMALS.          13 

leucocytes,  and  from  this  fact  we  may  assume  that  lactic  acid  is 
endowed  with  properties  that  are  antagonistic  to  those  of  bacterial 
products ;  in  other  words,  one  may  attribute  to  it  a  negative  chemio- 
tactic  power. 

Werigo  does  not  agree  with  this  interpretation.  It  seems  to  him 
that  lactic  acid  inhibits  the  attraction  of  leucocytes  not  by  exercising 
on  them  a  truly  repelling  action,  but  by  harming  them  or  paralyzing 
them,  and  so  preventing  them  from  feeling  the  attracting  influence 
of  bacterial  products.  Nor  does  Werigo  believe  in  the  repelling 
action  of  bacterial  secretions  any  more  than  he  does  in  the  same 
action  on  the  part  of  lactic  acid.  In  serious  infections  where  no 
phagocytosis  is  present  the  non-intervention  of  leucocytes  seems 
to  him  due  not  to  the  fact  that  they  are  repelled,  but  that  they  are 
simply  not  attractecj.  In  other  words,  there  is  an  absence  of  posi- 
tive chemiotaxis  without  any  actual  negative  chemiotaxis.  In 
brief,  "we  find,"  says  Werigo,  "  that  a  negative  chemiotaxis  for  leu- 
cocytes has  not  yet  been  proved  in  a  satisfactory  manner.  The 
question  must  be  left  to  further  experiments." 

Although  Werigo 's  objections  have  some  foundation,  we  believe 
that  a  negative  chemiotaxis  of  leucocytes  does  exist  and  that  it  may 
be  proved  experimentally. 

EXPERIMENT  1.  A  normal  guinea-pig  was  given  an  injection 
in  the  peritoneal  cavity  of  1  c.c.  of  a  two-day  culture  of  strepto- 
coccus. (The  culture  medium  is  a  mixture  of  bouillon  and  serum 
suitable  for  maintaining  the  virulence  of  the  organism.)  The 
streptococcus  employed  was  a  very  virulent  organism  kindly  fur- 
nished us  by  our  friend  Marmorek. 

Before  injection  the  peritoneal  cavity  was  found  to  contain  a  few 
leucocytes  largely  of  the  mononuclear  type  and  a  few  eosinophiles ; 
there  were  very  few  polynuclear  microphages. 

After  injection  a  small  amount  of  exudate  was  removed  at  inter- 
vals and  stained  preparations  were  made. 

Within  a  few  minutes  some  organisms  were  found  within  mononu- 
clear leucocytes.  But  as  these  leucocytes  are  few  in  number  a 
majority  of  the  organisms  remain  free. 

After  an  hour  the  polynuclear  leucocytes  are  to  be  found,  but  they 
are  still  few  in  number;  it  is  to  be  noted,  however,  that  the  majority 
of  them  have  taken  up  organisms.  The  number  of  these  leucocytes 


14  STUDIES  IN  IMMUNITY. 

then  increases  rapidly.  After  3  hours,  for  example,  it  is  found  that 
the  exudate  is  very  rich  in  polynuclears.  These  cells  are  far  more 
than  are  required  to  take  up  alkthe  organisms  present;  as  a  matter 
of  fact,  only  a  relatively  small  number  of  cocci  were  injected  and 
these  have  had  no  opportunity  to  increase.  But  no  matter  how 
numerous  the  phagocytes  are,  free  organisms  which  have  escaped  the 
destructive  phagocytic  action  are  still  found  in  the  surrounding  fluid. 
Only  a  certain  number  of  them  have  become  the  prey  of  the  white 
corpuscles. 

Then  a  second  phase  of  the  infection  begins.  The  number  of  micro- 
phages  continues  to  increase.  At  the  same  time  the  organisms  that 
have  not  been  taken  up  multiply  and  give  rise  to  new  individuals. 
At  the  end  of  6  hours  the  exudate  is  seen  to  contain  very  large 
numbers  of  phagocytes  and  at  the  same  time  a  considerable  num- 
ber of  organisms.  But  these  phagocytes  are  empty.  The  phago- 
cytosis of  the  coccus  noted  at  first  is  no  longer  present.  This  fact 
is  still  more  surprising  since  the  organisms  and  the  phagocytes  must 
still  be  in  contact;  it  would  seem  as  if  the  tactile  reaction  which 
the  cells  usually  show  would  suffice  to  produce  phagocytosis. 

It  must  be  that  some  active  influence  prevents  the  phagocytes 
from  taking  up  those  organisms  scattered  about  them.  What  is 
this  influence?  Two  explanations  occur:  (a)  either  the  leucocytes 
are  paralyzed  by  the  toxin  that  the  streptococcus  secretes  in  multi- 
plying; or,  (6)  the  leucocytes,  while  retaining  all  their  motility  and 
facility  for  taking  up  organisms,  may  have  been  subjected  to  a  nega- 
tive chemiotactic  influence  from  the  streptococcus  that  prevents 
them  from  taking  up  the  bacteria.  We  shall  see  that  the  first  of 
these  hypotheses  is  incorrect.  The  second  is  the  true  explanation. 

Eight  hours  after  injection,  when  the  animal  appears  to  be  very 
sick  and  the  peritoneal  exudate  is  alive  with  organisms  and  very 
rich  in  phagocytes,  we  give  a  second  injection.  We  inoculate  1  c.c. 
of  a  rich  bouillon  culture  of  Proteus  vulgaris  (24  hours  old);  this 
organism  is  only  slightly  virulent  and  is  easily  destroyed  by  phago- 
cytes. A  few  moments  later,  if  we  take  out  a  little  exudate,  we 
find  that  the  cells  which  refuse  to  take  up  the  streptococcus  have 
eagerly  taken  hold  of  the  new  organism  offered  them.  Within  a 
half  hour  almost  the  entire  culture  is  within  phagocytes.  It  is 
extraordinary  how  these  phagocytes  have  chosen  between  the  two 


STUDIES  ON  THE  SERUM  OF   VACCINATED  ANIMALS.          15 

varieties  of  bacteria.  With  greatest  delicacy  they  have  reacted  to 
a  new  chemical  substance  and  each  one  takes  up  numerous  Proteus 
organisms,  recognizable  by  their  rod  shape,  but  still  refuses  the 
streptococci  which  remain  scattered  throughout  the  preparation 
outside  the  cells.  The  protoplasm  of  the  phagocytes  acts  rapidly 
on  the  bodies  of  the  bacteria  that  have  been  engulfed.  These 
Proteus  bacilli  soon  manifest  changes  in  their  reaction  to  dyes:  a 
preparation  from  the  exudate  taken  a  short  time  after  the  second 
injection  or  kept  for  a  few  hours  in  a  moist  chamber  shows,  with  a 
contrast  stain  of  eosin  and  methylene  blue,  phagocytes  containing 
reddish  rods  (Proteus) ;  outside  the  cells  are  seen  numerous  strep- 
tococci stained  blue.  The  organisms  that  have  been  taken  up,  then, 
take  an  acid  stain  instead  of  the  usual  methylene  blue. 

We  may  conclude,  then,  that  the  leucocytes  of  the  peritoneal 
exudate  have  been  neither  killed  nor  paralyzed  by  a  toxin  from  the 
streptococcus.  Their  faculty  of  engulfing  has  remained  intact, 
but  they  refuse  to  enter  in  contact  with  the  streptococcus  owing 
to  the  fact  that  they  receive  a  negative  chemical  stimulation  from 
this  organism. 

It  may  be  clearly  seen  from  this  experiment  that  phagocytes  are 
capable  of  choosing  with  great  delicacy  between  organisms  offered 
them  on  account  of  their  reaction  to  chemical  substances.  It  is 
then  very  probable  that  when  an  animal  is  inoculated  with  a  given 
bacterium  the  phagocytes  first  take  hold  of  those  individual  or- 
ganisms that  are  most  attracting,  that  is  to  say,  the  least  dangerous. 
From  this  standpoint  it  is  easy  to  understand  that  the  increase  in 
virulence  by  passage  through  the  animal  body  is  due  at  least  in  part  to 
selection  on  the  part  of  the  phagocytes.  The  evolution  of  the  in- 
fection which  has  just  been  described  gives  evidence  of  this  fact. 
The  injection  of  the  relatively  scanty  culture  of  streptococcus 
caused  a  gathering  of  leucocytes  (it  is  known  that  the  injection  of 
simple  bouillon  without  the  presence  of  bacterial  products,  also 
causes  this  phenomenon).  Leucocytes  in  the  beginning  take  up  a 
certain  number  of  bacteria,  but  they  fail,  although  present  in  rela- 
tively larger  numbers  than  the  bacteria,  to  absorb  certain  individuals 
particularly  endowed  with  repelling  properties.  These  more  viru- 
lent organisms  may  multiply,  and  furnish  new  individuals  of  like 
virulence,  and  similarly  insusceptible  to  phagocytic  action. 


16  STUDIES  IN  IMMUNITY. 

We  believe,  then,  that  we  are  justified  in  repeating  what  we  wrote 
three  years  ago  on  the  increase  in  virulence  of  bacterial  infective 
agents:*  "When  we  inject  vibrios  subcutaneously  in  a  vaccinated 
guinea-pig  an  emigration  of  leucocytes  rapidly  takes  place.  The 
first  white  corpuscles  to  arrive  at  the  point  of  inoculation  find  a 
considerable  number  of  adversaries,  of  which  they  can  destroy  only 
a  few.  It  would  be  sufficient  for  a  few  of  these  vibrios  to  be  endowed 
with  a  slightly  more  intense  power  of  attraction  than  their  fellows, 
to  cause  the  phagocytes  to  direct  themselves  by  preference  toward 
these  organisms  and  to  take  them  up.  Very  slight,  almost  inappre- 
ciable differences  will  consequently  predestine  certain  bacteria  to 
rapid  phagocytosis.  In  the  same  way  an  inferiority  in  secretion  of 
toxic  products,  however  slight,  will  cause  a  predisposition  to  rapid 
destruction. 

"In  a  word,  leucocytes  kill  first  those  organisms  that  are  less 
resistant,  and  the  culture  inoculated  will  be  freed  first  of  those 
individuals  which  either  form  less  poison  than  their  fellows  or 
attract  the  leucocytes  more. 

"In  the  meantime  the  vibrios  that  have  been  left  alone  will  divide 
and  produce  new  organisms  which,  in  turn,  will  be  exposed  to  the 
attack  of  phagocytes.  These  latter  will  again  suppress  those  indi- 
viduals that  are  most  poorly  armed  for  the  struggle,  and  will  leave 
only  those  that  possess  in  highest  degree  the  two  characters  just 
noted.  Thanks  to  this  process  of  selection,  new  generations  of 
organisms,  like  those  represented  by  the  cultures  used  in  our  experi- 
ments, will  be  derived  for  the  most  part  from  those  bacteria  which 
have  been  endowed  with  certain  advantages." 

It  has  already  been  well  established  that  virulent  strains  of  an 
organism  attract  leucocytes  less  strongly  than  do  attenuated 
strains. 

This  phenomenon  of  selection  certainly  comes  into  play  in  mixed 
infections.  Phagocytes  may  direct  their  efforts  particularly  toward 
one  of  the  invaders  and  so  neglect  the  other. 

A  diversion  would  thus  be  caused  which  would  favor  the  develop- 
ment of  the  more  dangerous  infection.    As  we  have  given  a  very 
clear  experimental  example  of  this  fact,  it  may  be  assumed  that 
phenomena  of  the  same  sort  occur  under  natural  conditions. 
*  See  "Adaptative  changes  of  bacterial  cultures,  etc.,"  p.  6. 


STUDIES  ON  THE  SERUM  OF   VACCINATED  ANIMALS.          17 

In  animals  that  have  been  vaccinated  by  the  injection  of  cultures 
or  by  a  preventive  serum,  the  chemiotactic  response  of  leucocytes 
for  the  bacterium  with  which  the  immunization  has  been  brought 
about  has  been  increased.  The  number  of  phagocytes  is  in  general 
larger  in  immunized  animals  than  in  normal  animals :  the  army  of 
defenders  is  increased.  If  phagocytes  are  filled  up  by  injecting 
inert  powders  into  the  blood  stream,  or  if  the  animal  has  been 
weakened  by  fasting,  cold,  bleeding,  or  over-heating,  an  infection 
may  develop  which  under  normal  conditions  might  be  overcome. 
The  objections  that  have  been  directed  against  the  phagocytic 
theory  by  partisans  of  the  " bactericidal  theory  of  the  body  fluids" 
have  been  gradually  disposed  of.  The  body  fluids  do  not  always 
appear  to  be  of  importance  in  defending  the  animal  body.  It  has 
already  been  noted  that  bacteria  which  have  been  inoculated  in 
sera  may  suffer  simply  from  the  sudden  change  of  medium  to  which 
they  have  been  subjected.  This  is  particularly  true  when  they 
have  become  unaccustomed  to  living  in  body  fluids  on  account  of 
their  culture  on  artificial  media. 

Besides,  there  is  no  constant  or  necessary  parallel  in  normal 
animals,  any  more  than  in  vaccinated  animals,  between  the  anti- 
septic properties  of  serum  and  a  refractory  state.  It  is  true  that  the 
serum  of  animals  vaccinated  against  the  cholera  vibrio,  or  against 
the  vibrio  Metchnikovi,  is  endowed  with  an  energetic  bactericidal 
property  against  its  respective  organism,  while  the  serum  of  normal 
animals  has  only  very  weak  destructive  properties  for  these  bacteria. 
But  the  existence  of  a  strong  bactericidal  property  in  the  serum  of 
vaccinated  animals  is  exceptional  rather  than  usual.  Animals  well 
immunized  against  pneumonia,  tetanus,  hog-cholera,  diphtheria, 
etc.,  have  sera  endowed  with  preventive  or  curative  properties, 
which  show  no  bactericidal  property.  The  destruction  of  bacteria 
in  such  animals  is  always  brought  about  by  phagocytes. 

The  theory  of  the  " attenuation  of  bacteria  by  the  body  fluids" 
is  no  more  general  in  application  than  is  the  theory  of  the  "bac- 
tericidal property  of  the  body  fluids."  Organisms  cultivated  in 
the  serum  of  vaccinated  animals  generally  become  accustomed  to 
this  medium  and  suffer  no  diminution  in  their  pathogenic  power. 
No  diminution,  moreover,  is  produced  by  passage  through  a  vacci- 
nated animal;  on  the  contrary,  it  is  frequently  to  be  noted  under 


18  STUDIES  IN  IMMUNITY. 

these  conditions  that  an  increase  in  virulence  occurs,  due  apparently 
to  selection  by  the  phagocytes.*  Bacteria  that  develop  in  the  tissues 
of  refractory  animals  have  little  attraction  for  leucocytes,  which  is 
the  proof  of  their  pathogenic  power.  The  experiments  of  Charrin 
and  Roger  on  the  attenuation  of  Bacillus  pyocyaneus  are  too  open 
to  criticism  to  furnish  any  reasonable  data  for  the  doctrines  of  an 
attentuation  by  body  fluids. 

Since  the  intervention  of  phagocytes  is  regularly  observed  in 
animals  that  defend  themselves  against  invading  organisms,  the 
phagocytic  theory  is  fully  capable  of  explaining  the  problem  of 
immunity.  But  although  the  fact  that  phagocytes  are  the  usual 
factors  in  immunity  is  undeniable,  their  importance  in  certain 
special  cases  may  be  limited.  These  limitations  have  been  found 
particularly  in  infections  caused  by  organisms  belonging  to  the 
group  of  vibrios;  and  it  is  particularly  in  regard  to  the  cholera 
vibrio  that  the  discussion  between  the  phagocytic  doctrine  and  the 
"humoral"  theory  has  grown  very  active.  There  is,  in  fact,  in 
these  special  cases  an  evident  correlation  between  the  appearance 
of  a  bactericidal  power  in  serum  and  the  beginning  of  the  refractory 
condition.  This  destructive  property,  though  very  slight  in  normal 
animals,  becomes  very  marked  in  vaccinated  animals,  and  if  it 
should  be  shown  in  such  animals  that  the  destructive  properties 
of  the  serum  are  present  during  life,  the  intervention  of  phagocytes 
would  no  longer  appear  necessary  in  dissipating  the  infection;  this 
point,  however,  is  one  that  has  never  been  demonstrated.  The 
researches  of  various  observers,  and  particularly  of  Metchnikoff, 
tend  to  convince  us  that  the  tissue  fluids  possess  during  life  no 
bactericidal  property  comparable  to  that  shown  by  the  serum  in 
vitro.j 

One  of  the  principal  objects  of  the  present  study  is  to  deter- 
mine whether  these  bactericidal  substances  are  not  present  in  cells 
in  the  living  body.  It  is  to  be  determined  whether  it  is  the  leuco- 
cytes particularly  that  contain  the  bactericidal  substances,  whether 
the  elaboration  of  these  substances  is  a  special  manifestation  of 
their  protective  activity,  and  whether  this  activity  is  brought  about 

*  See  p.  4. 

t  See  Metchnikoff,  Etudes  sur  I'immunite",  4  e  memoire.  Ann.  de  1'Institut 
Pasteur,  1891. 


STUDIES  ON   THE   SERUM  OF  VACCINATED  ANIMALS.        19 

by  an  adaptation  that  occurs  in  them  owing  to  vaccination.  If 
such  were  the  origin  of  these  substances,  the  appearance  of  a  bac- 
tericidal power  in  the  serum  of  vaccinated  animals  would  be  indic- 
ative only  of  the  degree  of  perfection  which  phagocytosis  has 
reached. 

Whatever  may  be  the  degree  of  bactericidal  activity  in  animals 
vaccinated  against  cholera,  phagocytosis  can  no  longer  be  con- 
sidered as  a  phenomenon  of  secondary  importance  in  the  destruction 
of  this  organism.*  It  will  be  found,  to  be  sure,  as  Pfeiffer  f  has 
recently  shown,  that  vibrios  injected  into  the  peritoneal  cavity  of  a 
vaccinated  animal  (where  leucocytes  are  always  present)  may  be 
for  the  most  part  destroyed  by  the  fluid  without  having  been  taken 
up  by  phagocytes,  but  it  is  none  the  less  true  that  many  of  them 
are  taken  up  in  the  manner  that  Metchnikoff  has  demonstrated. 
Metchnikoff  injected  a  culture  of  cholera  into  the  peritoneal  cavity 
of  a  vaccinated  animal,  and  then  withdrew  after  a  certain  time  a 
small  amount  of  exudate,  in  which  phagocytes  containing  vibrios 
are  to  be  seen.  If  the  exudate  is  placed  in  a  moist  chamber  at  body 
temperature,  it  is  found  that  phagocytes  removed  in  this  manner 
from  the  animal  body,  and  subjected  to  unfavorable  conditions,  no 
longer  successfully  oppose  the  development  of  the  living  organisms 
that  they  have  taken  up;  their  growth  goes  on  inside  the  cells, 
which  soon  become  veritable  sacs  stuffed  with  vibrios  that  finally 
burst  and  let  the  organisms  escape  into  the  surrounding  fluid. 

Pfeiffer  does  not  consider  these  facts  of  great  importance.  Let  us 
take  his  point  of  view  for  a  moment  and  admit  that  the  phenomena 
of  phagocytosis  have  only  a  secondary  function  and  that  the 
destruction  of  the  vibrio  in  the  vaccinated  animal  is  due  primarily  to 
the  activity  of  the  body  fluids  apart  from  cellular  activity;  it  would 
be  reasonable  to  conclude,  then,  that  this  humoral  action  would  occur 
with  greater  intensity  in  those  parts  of  the  body  most  richly  endowed 
with  bactericidal  substances  irrespective  of  the  presence  of  phago- 
cytes. If  the  blood  of  a  rabbit  or  guinea-pig  immunized  against 
cholera  contains  more  of  the  destructive  substances  than  the  peri- 
toneal exudate,  it  is  reasonable  to  conclude  that  it  is  in  the  blood 

*  See  Cantacuzene,  Recherches  stir  le  mode  de  destruction  du  vibrion  choleVique 
dans  1'organisme,  1894. 

t  Pfeiffer,  Zeitschrift  fur  Hygiene,  1894,  XVIII,  p.  1. 


20  STUDIES  IN  IMMUNITY. 

that  the  least  phagocytosis  takes  place  and  that  here  especially 
the  vibrio  will  be  killed  and  disappear  entirely,  apart  from  any 
intervention  of  the  cellular  protoplasm. 

It  may  indeed  be  shown  that  the  serum  of  a  rabbit  vaccinated 
against  cholera  is  more  bactericidal  than  its  peritoneal  exudate. 

Before  considering  the  experiments  that  we  have  performed  on 
this  subject,  it  might  be  well  to  recall  briefly  the  method  of  deter- 
mining the  relative  bactericidal  power  of  body  fluids  and  the  means 
of  ascertaining  which  of  two  fluids  —  or  of  two  sera,  to  be  exact  — 
is  the  more  destructive  for  a  given  micro-organism. 

Small  carefully  measured  amounts  of  the  fluids  to  be  compared 
(1  c.c.  for  example)  are  placed  in  separate  test  tubes.  The  growth 
of  the  organism,  which,  let  us  say,  is  a  culture  of  cholera  on  agar,  is 
suspended  in  a  few  cubic  centimeters  of  salt  solution  (0.6  per  cent) 
or  of  bouillon.  The  first  of  the  sera  is  inoculated  with  a  platinum 
loop  of  this  culture  dilution.  The  loop  is  agitated  in  the  fluid  with 
extreme  precaution  to  distribute  uniformly  all  the  bacteria  intro- 
duced; this  serum  has  then  become  a  homogeneous  medium  con- 
taining the  organisms.  A  small  amount  of  this  inoculated  serum 
is  then  carefully  withdrawn  by  means  of  the  loop  and  carried  over 
into  a  tube  containing  fluid  gelatin  at  body  temperature.  The 
gelatin  is  then  shaken  to  scatter  the  inoculated  organisms,  and 
poured  into  a  Petri  dish.  (First  culture.) 

The  number  of  colonies  that  develop  indicates  relatively  exactly 
the  number  of  bacteria  contained  in  a  loop  of  the  inoculated  serum. 
Since  the  operation  has  been  carried  out  rapidly,  the  micro-organisms 
carried  over  to  the  gelatin  have  been  in  contact  with  the  serum  for 
a  short  time  only,  and  whatever  bactericidal  power  the  serum  may 
possess  has  not  had  sufficient  time  to  affect  them.  After  a  certain 
time  the  same  amount  of  serum,  measured  accurately  by  means  of 
the  same  loop,  is  again  withdrawn.  A  second  gelatin  plate  is  inocu- 
lated. (Second  culture.)  If  the  serum  is  bactericidal,  this  second 
plate  should  contain  either  less  colonies  than  the  first,  or  perhaps 
none  at  all.  If  we  repeat  successively  at  intervals  the  same 
procedure,  we  shall  have  gelatin  plates  that  will  give  us  an  indi- 
cation of  the  number  of  micro-organisms  remaining  alive  in  the 
serum. 

If  the  same  set  of  manoeuvres  is  performed  in  an  identical  manner 


STUDIES   ON   THE   SERUM   OF    VACCINATED  ANIMALS.        21 


at  the  same  time  with  two  more  or  less  completely  bactericidal 
liquids,  a  comparison  may  be  drawn  as  to  their  bactericidal  value 
according  to  the  number  of  colonies  that  grow  on  the  respective 
plates.  It  is  obvious  that  the  same  emulsion  of  organisms  is  used 
for  each  series  of  tubes,  and  the  same  platinum  loop  is  used  so  as  to 
render  the  quantities  of  liquids  strictly  comparable.  A  table  may 
be  made  indicating  the  number  of  colonies  which  have  developed 
on  each  plate  and  the  time  after  inoculation  at  which  the  trans- 
plantations were  made.  With  this  explanation  we  offer  the  protocol 
of  an  experiment  made  for  the  purpose  of  comparing  the  bactericidal 
properties  of  the  blood  and  of  the  peritoneal  exudate  from  an  animal 
vaccinated  against  cholera. 

EXPERIMENT  2.  A  rabbit  weighing  1720  grams  had  been  well 
vaccinated  against  the  vibrio  of  Massaouah;  two  weeks  after  the 
last  injection  a  small  amount  of  blood  was  drawn,  from  which  was 
obtained  serum  I;  two  sponges  previously  washed  and  boiled  in 
water,  sterilized,  and  dried  (June  14,  1894),  were  then  placed  in  the 
peritoneal  cavity.  They  were  removed  the  next  day  and  found 
to  be  filled  with  fluid  that  was  squeezed  into  two  sterile  vessels. 
(Exudates  I  and  II.)  The  leucocytes  in  this  fluid  were  infrequent 
(about  500  to  the  c.  mm.)  and  quite  a  number  of  them  adhered  to 
the  sponge.  A  second  specimen  of  blood  was  then  taken  (serum  II) 
which  was  found  to  contain  8200  leucocytes.  One  cubic  centimeter 
of  each  of  the  samples  of  serum  and  of  the  peritoneal  exudates  was 
placed  in  a  tube  and  their  respective  bactericidal  powers  against  the 
vibrio  of  Massaouah  determined  by  the  plate  method.  Since  it  had 
already  been  determined  that  the  serum  was  rather  strongly  bac- 
tericidal, a  relatively  large  amount  of  this  culture  was  planted  each 
time  (a  loop  of  a  vigorous  agar  culture,  24  hours  old,  suspended  in 
5  c.c.  of  salt  solution  of  0.6  per  cent).  The  following  plates  were 
made  at  intervals  indicated  in  the  table: 

NUMBER  OF  COLONIES. 


Time  of  cultures. 

Serum  I. 

Serum  II. 

Peritoneal  Exu- 
date I. 

Peritoneal  Exu- 
date II. 

I. 

II. 
III. 

June  15,  1894,  5 
June  15,  1894,  7 
June  16,  1894,  9 

P.M. 
P.M. 
30  A.M. 

25,200 
0 
0 

23,400 
0 
0 

24,000 
35 
Innumerable 

22,200 
20 
Innumerable 

22  STUDIES  IN  IMMUNITY. 

The  fluid  portion  of  the  blood  contains,  then,  more  bactericidal 
substance  than  an  equal  amount  of  the  peritoneal  exudate.  Be- 
sides this,  the  total  volume  of  blood  is  far  greater  than  that  of  the 
peritoneal  fluid  and  consequently  contains  very  much  more  of  this 
destructive  substance.  Vibrios  introduced  directly  into  the  circu- 
lation and  disseminated,  are  therefore  exposed  to  the  maximum 
bactericidal  action.  If  the  phagocytes  are  not  indispensable  agents 
in  the  destruction  of  the  vibrio  inoculated  into  the  peritoneum 
they  must  be  still  less  important  factors  in  the  destruction  of 
organisms  injected  into  the  circulation.  Phagocytosis,  however, 
under  these  latter  conditions  occurs  energetically  and  rapidly. 

EXPERIMENT  3.  (a)  A  guinea-pig  weighing  420  grams  had  been 
well  vaccinated  against  the  true  cholera  vibrio  (a  culture  labelled 
Eastern  Prussia).  One-third  of  a  24-hour  agar  culture  of  this 
organism  suspended  in  0.6  per  cent  salt  solution  was  injected  into 
the  jugular  vein.  At  intervals  a  drop  of  blood  was  taken  either 
from  the  paw  or  from  the  ear  and  spread  on  slides.  After  a  quar- 
ter of  an  hour  the  animal  was  killed  and  preparations  were  made 
from  the  liver,  spleen  and  heart's  blood.  The  preparations  were 
fixed  by  5  per  cent  carbolic  acid  solution  instead  of  heat,  and 
stained  by  Ehrlich's  method  (eosin  and  methylene  blue). 

Preparations  of  blood  taken  at  4,  5,  9  and  13  minutes  after  injec- 
tion show  phagocytes  containing  vibrios.  These  phagocytes  are 
particularly  numerous  in  specimens  taken  very  soon  (4  or  5  minutes) ; 
one  can  see  well  stained  and  perfectly  intact  vibrios  within  the 
protoplasm.  In  the  specimens  taken  9  to  13  minutes  after  injec- 
tions, and  particularly  in  the  smears  from  the  liver  and  kidneys, 
the  phagocytes  contain,  in  addition  to  the  normal  vibrios,  incom- 
pletely or  irregularly  stained  organisms  showing  definite  granules. 
A  culture  from  the  heart's  blood  on  agar  gave  a  few  colonies. 
Cultures  from  the  liver  and  kidney  contained  a  great  many  more 
colonies. 

(b)  A  well  vaccinated  guinea-pig  of  530  grams  weight  (V.  cholerse, 
Eastern  Prussia)  was  given  a  third  of  a  culture  into  the  jugular. 
The  animal  was  killed  one  half  hour  after  injection;  the  number  of 
leucocytes  had  fallen  from  16,500  to  8000.  Phagocytosis  is  evident 
in  blood  drawn  5,  8,  and  12  minutes  after  injection.  Many  poly- 
nuclears  in  smears  from  the  spleen  and  lung.  The  leucocytes  of  the 


STUDIES   ON  THE  SERUM  OF  VACCINATED  ANIMALS.        23 

spleen  contain  irregular  vibrios  which  are  stained  in  places,  and  of  a 
reddish  or  violet  tinge;  this  evidences  the  chemical  alterations  that 
these  vibrios  have  undergone.  In  the  heart's  blood  half  an  hour 
after  injection  there  are  a  few  phagocytes  containing  intact  and 
well  colored  vibrios.  A  culture  from  the  heart  on  agar  showed  a 
few  colonies ;  cultures  from  the  liver  and  especially  from  the  spleen 
were  rich  in  organisms. 

(c)  A  half  culture  injected  into  the  jugular  of  an  immunized  guinea- 
pig  (480  grams).  The  dose  was  so  large  that  the  animal  died  in 
4  minutes  with  symptoms  of  asphyxia.  It  is  well  shown,  however, 
in  preparations  from  the  blood  taken  at  death  that  phagocytosis 
was  already  present. 

When  the  number  of  bacteria  injected  into  the  circulation  is  not 
too  large  the  blood  soon  becomes  sterile,  but  the  organs  still  contain 
living  vibrios  that  grow  well  on  agar.*  The  number  of  leucocytes 
in  the  blood  is  decreased  during  this  same  period.  The  phenomena 
following  injection  of  cholera  vibrios  into  the  circulation  are  quite 
similar,  then,  to  those  obtained  under  the  same  conditions  with  other 
pathogenic  micro-organisms]  the  infecting  organisms  are  taken  up  by 
the  phagocytes  and  carried  by  them  to  the  internal  organs,  where  they 
multiply.  Immunity  against  cholera  is  dependent,  then,  on  the 
same  mechanism  as  immunity  against  other  infections. 

Phagocytic  activity  is  apparently  not  easily  inhibited,  since  the 
accumulation  of  the  organism  in  the  internal  organs  and  the  dis- 
appearance of  leucocytes  from  the  blood  may  be  noted  even  in 
chloroformed  animals.  Chloroform  anesthesia  then,  sufficient  to 
deaden  the  nervous  centers,  has  no  effect  on  the  activity  of  phag- 
ocytes. 

EXPERIMENT  4.  Vaccinated  guinea-pig  "A,"  weight  400  grams; 
the  animal  was  well  anesthetized,  so  that  all  reflexes  were  lost. 
Five  minutes  later  one-sixth  of  a  culture  of  the  vibrio  from  Eastern 
Prussia  was  injected  into  the  jugular.  The  anesthetic  was  con- 
tinued; the  animal  died  after  20  minutes,  as  too  much  chloroform 
was  given.  The  leucocyte  count  had  fallen  from  13,000  to  4300. 
The  heart's  blood  was  sterile,  whereas  the  extract  from  liver,  spleen 
and  lungs  gave  positive  cultures. 

*  Vibrios  that  have  been  in  contact  with  bactericidal  substances  frequently 
fail  to  grow  in  gelatin,  although  they  still  grow  in  agar. 


24  STUDIES   IN   IMMUNITY. 

A  control  guinea-pig  "B,"  weighing  400  grams,  had  been  vacci- 
nated in  the  same  manner  as  "A."  One-sixth  of  a  culture  was 
given  this  animal  without  chloroform.  In  20  minutes  the  leucocyte 
count  had  fallen  from  9500  to  5000.  At  this  time  the  blood  was 
sterile;  liver,  lung  and  spleen  gave  positive  cultures. 

II.   LEUCOCYTES  AND  THE  BACTERICIDAL  POWER  OF  SERUM. 

Phagocytosis  is  not  of  secondary  importance  in  the  mechanism 
of  cholera  immunity.  Leucocytes  take  up  vibrios  which  soon  show 
alterations  in  form  and  reaction  to  dyes  within  the  protoplasm, 
which  indicates  that  they  have  been  affected  by  some  harmful 
substance.  Is  it  not  possible  that  the  bactericidal  substance  in 
serum  comes  from  the  leucocytes?  Metchnikoff  has  already  offered 
the  suggestion  (1887)  that  the  bactericidal  substances  of  serum 
might  be  of  leucocytic  origin.  In  1899  he  wrote:*  "I  might  add 
that  those  who  have  asserted  that  the  action  of  serum  against 
bacteria  is  independent  of  leucocytes  have  not  taken  into  considera- 
tion those  substances  liberated  in  the  serum  as  the  result  of  the 
destruction  of  leucocytes.  It  has  been  repeatedly  noticed  that 
these  cells  when  removed  from  their  normal  surroundings  break 
up  and  liberate  their  contents  into  the  surrounding  fluid."  Hankinf 
and  an  English  experimenter,  Kanthack,  later  attributed  to 
the  eosinophiles  the  secretion  of  bactericidal  substances.  Accord- 
ing to  this  conception  these  latter  cells  may  be  broken  up  in  the 
blood  stream  and  so  affect  the  micro-organisms  that  have  entered 
the  body.  Bacteria,  killed  or  attenuated  by  contact  with  these 
substances,  might  later  on  be  taken  up  by  phagocytes.  According 
to  this  conception  also  it  is  the  phagocytes  which  form  the  bacteri- 
cidal substances.  The  engulfing  of  bacteria  by  living  cells  instead 
of  being  a  phenomenon  essential  to  the  defense  of  the  organism 
would  be  rather  an  accessory  phenomenon  subsequent  to  the 
destruction  of  the  bacteria  by  bactericidal  substances  dissolved  in 
the  plasma. 

*  Annales  de  1'Institut  Pasteur,  December,  1889. 

t  Hankin,  Ueber  den  Ursprung  und  Vorkommen  von  Alexinen  im  Organismus 
(Centralblatt  fur  Bakteriologie,  12,  Nos.  22  et  23,  December,  1892);  Ueber  die 
Theorie  der  Alexocyten  (Centralblatt  fur  Bakteriologie,  14,  No.  25,  December, 
1893). 


STUDIES  ON   THE   SERUM   OF    VACCINATED   ANIMALS.        25 

Hankin's  theory,  although  resembling  the  bactericidal  theory  of 
body  fluids,  is  not  open  to  the  objections  that  may  be  offered  to  the 
latter. 

The  two  essential  points  in  these  doctrines  are  the  following: 
1.  There  is  a  bactericidal  substance  in  solution  in  the  body  fluids, 
and  bacteria  are  taken  up  by  the  phagocytes  only  after  having  been 
subjected  to  the  influence  of  this  substance.  But,  far  from  being 
subsequent  to  a  preparing  action  on  the  part  of  the  bactericidal 
substance,  phagocytosis  takes  place  with  surprising  rapidity;  it 
begins,  indeed,  immediately  after  inoculation  of  the  organisms.  In 
the  case  of  cholera  particularly  we  have  just  seen  that  shortly  after 
the  intravenous  injection  of  cholera  vibrios  a  number  of  them  are 
found  inside  of  phagocytes.  And,  what  is  more,  it  has  frequently 
been  proved  by  Metchnikoff  that  cells  can  take  up  living  bacteria. 

2.  Bactericidal  substances  are  elaborated  by  eosinophilic  leuco- 
cytes. It  is  known,  however,  that  these  cells  are  of  no  great  impor- 
tance in  defense  of  the  organism.  Phagocytosis  and  the  destruction 
of  bacteria,  moreover,  goes  on  very  well  in  invertebrates,  although 
they  have  no  eosinophiles.  As  we  shall  see  later  on  in  our  experi- 
ments on  "phagocytosis  in  vitro,"  various  bacteria  when  mixed 
with  an  exudate  rich  in  phagocytes,  but  containing  no  true  eosino- 
philes, are  rapidly  taken  up  and  modified  in  their  form  and  staining 
reaction. 

Denys  *  and  his  pupils  think  that  leucocytes  play  an  important 
part  in  the  elaboration  of  bactericidal  substances;  and  they  en- 
deavor to  reconcile  the  phagocytic  and  the  humoral  theories. 
Buchner  f  is  inclined  to  admit  that  leucocytes  are  of  great  impor- 
tance in  the  formation  of  bactericidal  substances. 

There  are  two  phases  to  the  problem;  first:  do  the  leucocytes 
form  the  bactericidal  substances  found  in  serum?  And,  secondly: 
if  the  leucocytes  really  form  these  bactericidal  substances,  do  they 
retain  them  during  life  or  do  they  excrete  them  into  the  surrounding 
plasma? 

It  is  perfectly  evident  that  in  order  to  draw  conclusions  from 
experiments  which  are  of  any  value  to  our  knowledge  of  immunity, 
we  must  study  the  bactericidal  property  in  its  simplest  aspects  and 

*  La  Cellule,  1893  et  1894. 

f  Buchner,  Munchener  medic inische  Wochenschrift,  1894. 


26 


STUDIES  IN   IMMUNITY. 


when  it  bears  some  definite  relation  to  a  condition  of  resistance.  It 
often  happens,  as  we  know,  that  the  serum  of  an  animal  is  bacteri- 
cidal for  a  given  micro-organism,  although  the  animal  itself  is  quite 
susceptible  to  infection  by  that  very  organism.  For  example, 
rabbit  serum  is  bactericidal  for  the  anthrax  bacillus,  although  the 
rabbit  is  highly  susceptible  to  this  bacterium.* 

This  is  simply  an  example  of  the  spontaneous  bactericidal  power 
found  in  normal  animals;  its  significance  from  the  standpoint  of 
immunity  is  not  clearly  established,  and  its  importance  is  doubtless 
exaggerated. 

We  shall  consider  then,  more  particularly  the  strong  bacteri- 
cidal property  in  animals  immunized  against  certain  infections, 
for  example,  against  cholera.  The  bactericidal  property  of  the 
serum  of  normal  animals  against  the  cholera  vibrio  is  insignificant. 
Following  vaccination  the  bactericidal  property  becomes  very 
distinct.  Since  it  is  very  evident  during  the  stage  when  the 
animal  is  insusceptible  to  infection,  its  relation  to  immunity  is 
unmistakable. 

*  The  aqueous  humor  of  the  rabbit  is  bactericidal  for  anthrax.  This  fluid 
contains  few  or  no  leucocytes;  and  it  would  seem,  therefore,  difficult  to  attribute 
the  bactericidal  property  to  cells.  But  it  is  to  be  noted  that  bacteriolysis  occurs 
in  this  instance  in  a  highly  susceptible  animal,  and  obviously,  therefore,  bears  no 
relation  to  a  condition  of  resistance.  It  differs,  also  essentially,  from  the  bacte- 
riolysis that  occurs  in  certain  instances  of  immunity,  as  the  property  in  this  in- 
stance resists  heating,  to  60  degrees  for  an  hour.  The  bactericidal  property  would 
seem  to  be  more  like  that  shown  by  such  liquids  as  bouillon. 

It  is  evident  from  the  following  table  that  the  aqueous  humor  of  the  rabbit 
even  after  heating  for  an  hour  to  60°  C.  is  quite  bactericidal  for  B.  anthracis. 

EXPERIMENT  4.  Aqueous  humor  was  taken  from  a  rabbit.  Part  of  it  was 
heated  to  60  degrees  for  an  hour,  and  the  rest  was  not  heated.  Equal  amounts 
of  each  fluid  were  placed  in  tubes.  A  culture  of  non-spore-forming  anthrax 
bacilli,  24  hours  old,  was  suspended  in  0.6  per  cent  NaCl  solution  and  planted  in 
these  tubes.  Gelatin  plates  were  made  at  intervals. 

NUMBER  OF  COLONIES. 


Time  of  cultures. 

Aqueous  humor  60°. 

Unheated  Aqueous 
humor. 

T 

May  12,  1895,  1.30  P.M  

1,200 

1,400 

IT 

May  12    1895   2        p  M 

16 

44 

TTT 

May  12,  1895,  4.15  P.M  

13 

9 

IV. 

May  13,  1895,  11      A.M  

0 

0 

STUDIES  ON  THE   SERUM  OF   VACCINATED  ANIMALS.         27 

Our  experiments  have  been  carried  out  largely  with  two  cholera 
vibrios:  the  vibrio  of  Massaouah,  isolated  by  Pasquale,  and  the 
vibrio  from  Eastern  Prussia.  We  shall  not  consider  here  whether 
or  not  the  vibrio  of  Massaouah  is  of  the  same  variety  as  the  typical 
Koch  vibrio.  The  researches  of  Pfeiffer  would  indicate  that  there 
are  characteristic  differences  between  cholera  vibrios  from  different 
sources,  which  would  lead  us  to  think  that  they  do  not  all  belong  to 
the  same  strain  of  bacteria.  It  would  appear  that  there  are  several 
varieties  of  cholera  vibrios  and  that  one  of  these  varieties  which  is 
widely  distributed  and  is  of  great  importance  is  represented  by 
Koch's  vibrio.  This  organism,  according  to  Pasquale,  is  the  sole 
cause  of  epidemics.  However  that  may  be,  animals  immunized 
against  the  vibrio  of  Massaouah  have  energetic  specific  pre- 
ventive and  bactericidal  properties  ijn  their  sera  and  therefore 
fulfill  the  conditions  we  desire.  What  is  more,  this  vibrio  has  the 
advantage  of  being  highly  virulent  and  relatively  constant  in  its 
pathogenicity. 

We  have  tried  to  effect  a  separation  between  the  blood  fluid  and 
the  blood  cells  in  an  animal  vaccinated  against  cholera.  For  this 
purpose  it  was  necessary  to  obtain  a  more  or  less  cell-free  plasma, 
and  we  succeeded  in  doing  this  by  causing  an  edema  of  the  leg  or 
ear  by  venous  compression.  The  edema  fluid  represents  blood 
plasma  filtered  under  pressure  and  almost  completely  deprived  of 
cells.  The  walls  of  blood  vessels  are  not  sufficiently  impermeable 
to  hold  back  all  the  cells,  and  the  fluid  obtained  in  this  manner  con- 
tains a  few  red  blood  cells  and  very  infrequent  leucocytes.  A  com- 
parison may  be  made  between  fluid  obtained  in  this  manner  and 
the  serum  of  the  same  animal  as  regards  their  respective  bactericidal 
powers.  The  serum  is  obtained  by  spontaneous  coagulation  of  the 
whole  blood  containing  many  cells.  If  during  life  bactericidal 
substances  remain  within  the  cells  and  appear  in  the  blood  fluid 
only  after  removal  from  the  body  and  coagulation,  a  distinct  dif- 
ference should  appear  between  these  two  fluids. 

EXPERIMENT  5.  A  rabbit  weighing  1790  grams  was  vaccinated 
against  the  Vibrio  Massaouah.  The  serum  of  this  rabbit  was  tested 
for  bactericidal  and  preventive  properties  and  found  to  be  markedly 
bactericidal.  One-fifth  of  a  cubic  centimeter  moreover  protected 


28 


STUDIES  IN   IMMUNITY. 


against  iV  of  a  24-hour  culture,  a  fatal  dose,  and  even  against  TV  of  a 
culture. 

A  few  days  later  an  edema  was  produced  in  this  rabbit  by  con- 
stricting the  root  of  the  ear  with  a  rubber  ring  (edema  fluid  UA"). 

One  of  the  fore  legs  received  the  same  treatment,  and,  on  tapping, 
a  clear  liquid,  edema  fluid  "B,"  was  obtained,  which  was  slightly 
pinkish  but  contained  very  few  leucocytes  (one  to  two  hundred  per 
cubic  mm.).  A  few  cubic  centimeters  of  blood  were  then  taken  and 
found  to  contain  3600  leucocytes.  The  serum  of  a  normal  rabbit 
was  used  as  a  control.  One  cubic  centimeter  of  each  of  these  fluids 
was  put  in  a  separate  tube.  A  platinum  loop  from  a  24-hour 
culture  of  the  organism  suspended  in  5  c.c.  of  salt  solution  was 
placed  in  each  tube. 

EXPERIMENT  WITH  EDEMA   "A"   (FROM    EAR). 


Time  of  cultures. 

Serum  of  vac- 
cinated ani- 
mal. 

Edema  of  vac- 
cinated ani- 
mal. 

Serum  of 
normal  rab- 
bit. 

T 

April  27,  1894,  5       P.M  

15,000 

14,000 

12,900 

TT 

April  27,  1894,  5.30  P.M  

1 

6 

840 

TTT 

April  27,  1894,  6.30  P.M  

0 

3 

180 

TV 

April  27    1894   9       P  M 

o 

o 

420 

v 

April  28    1894   9  15  A.M. 

o 

Innumerable 

Innumerable 

EXPERIMENT  WITH  EDEMA   "B"    (FROM  LEG). 


T 

April  29, 

1894, 

6       P 

M  

18,900 

20,000 

24,000 

TT 

April  29, 

1894, 

6.30  P 

M  

0 

7 

2,100 

III. 

April  29, 

1894, 

7.45  P 

M  

0 

0 

780 

IV. 

April  29, 

1894, 

10.30  P 

M  

0 

2 

1.200 

V. 

April  30, 

1894, 

10.30  A 

M  

0 

Innumerable 

Innumerable 

The  day  following  inoculation  the  tubes  containing  normal  rabbit 
serum  and  edema  fluid  were  both  cloudy  and  filled  with  vibrios; 
the  serum  of  the  vaccinated  rabbit  was  perfectly  clear.  Two  days 
later  a  culture  made  on  agar  from  this  serum  showed  that  it  had 
remained  sterile.  The  difference  in  bactericidal  power  between 
the  edema  and  the  serum  of  the  vaccinated  animal  is  therefore 
evident. 


STUDIES  ON  THE    SERUM  OF  VACCINATED    ANIMALS.        29 


Although  the  cholera  vibrio  grew  well  in  the  edema  fluid,  this  fluid 
showed,  during  the  first  few  hours,  a  very  distinctive  bactericidal 
property,  although  less  than  that  of  serum.  The  normal  serum  also 
possessed  a  certain  bactericidal  property.  It  is  to  be  noted  that 
the  gelatin  plate  made  three  or  four  hours  after  inoculation  from 
the  edema  fluid  was  sterile.  It  is  evident,  however,  that  not  all  the 
organisms  in  this  fluid  were  killed,  since  the  cultures  made  on  the 
following  day  showed  numerous  colonies.  It  should  be  remarked 
in  this  connection  that  gelatin  is  not  a  very  favorable  medium  on 
which  to  grow  the  cholera  vibrio.  Vibrios  that  are  slightly  weakened 
but  are  still  alive  may  fail  to  grow  on  gelatin  when  they  grow  very 
well  on  agar.  We  have  often  noted  this  fact,  and  a  growth  on  agar, 
then,  must  be  considered  the  necessary  criterion  of  the  sterility  of  a 
sample  of  inoculated  serum. 

EXPERIMENT  6.  A  rubber  ring  was  placed  about  the  fore  leg  of 
a  guinea-pig  vaccinated  by  sterilized  and  living  cultures  of  the 
cholera  vibrio  from  Eastern  Prussia.  An  edema  was  formed  which 
was  withdrawn  and  found  to  be  slightly  reddish  in  color  and  to 
contain  a  few  leucocytes.  A  specimen  of  blood  was  taken  contain- 
ing 11,000  leucocytes  per  cubic  centimeter.  The  serum  and  the 
edema  fluid  were  inoculated  with  a  24-hour  culture  of  the  vibrio 
(Oriental  Prussia). 

NUMBER  OF  COLONIES. 


Time  of  cultures. 

Serum. 

Edema. 

I 

February  23    1895    11 

A  M 

15,600 

15,000 

II 

February  23    1895     1 

30  P  M. 

0 

0 

III. 

February  23,  1895,    6 

P.M  

0 

660 

IV. 

February  24,  1895,  10 

A.M  

0 

Innumerable 

The  edema  fluid  was  cloudy  the  next  day,  and  under  the  micro- 
scope an  enormous  number  of  vibrios  were  found  in  it;  the  serum 
was  clear  and  contained  no  bacteria.  The  edema  fluid,  however, 
had  sufficient  activity  to  weaken  the  vibrios  during  the  first  few 
hours  and  to  prevent  growth  on  gelatin.  The  serum  gave  no 
growth  on  agar. 

The  production  of  edema  is  the  best  means  of  separating  the  cells 
from  the  blood  plasma,  but  there  is  another  method  which  may  be 


30  STUDIES  IN  IMMUNITY. 

used  to  study  the  effect  of  a  variation  in  leucocytes  on  the  bacteri- 
cidal property  of  the  blood.  This  method  consists  in  artificially 
lowering  the  number  of  leucocytes  in  the  circulating  blood  and 
testing  the  bactericidal  property  of  the  blood  before  and  after. 

The  most  efficient  method  of  lowering  the  number  of  leucocytes 
is  to  inject  bacteria,  but  the  method  could  not  be  used  in  this  work, 
as  the  introduction  of  bacterial  substances  might  affect  the  bacteri- 
cidal property  of  the  blood  in  some  manner.  Instead  of  bacteria, 
then,  an  emulsion  of  some  fine  inert  powder  such  as  carmin  must  be 
used.  It  has  been  asserted  that  cooling  an  animal  in  water  brings 
about  a  fall  in  the  number  of  leucocytes,  but  we  have  had  no  success 
with  this  method.  The  injection  of  carmin  generally  produces  a 
rapid  hypoleucocytosis.  In  certain  cases,  however,  the  fall  in 
white  corpuscles  does  not  follow.  It  is  probable  that  the  taking 
up  of  grains  of  carmin  by  phagocytes  depends  on  the  tactile 
reaction  of  these  cells.  It  is  important  to  determine  that  the 
fluid  in  which  the  carmin  is  suspended  has  no  positive  chemio- 
tactic  influence  on  leucocytes,  as  shown  by  an  experiment  with 
capillary  tubes. 

EXPERIMENT  7.  A  guinea-pig  weighing  355  grams  had  been 
vaccinated  against  the  vibrio  of  Massaouah.  A  small  amount  of 
blood  was  taken  from  the  animal  and  found  to  contain  11,000 
leucocytes.  One-tenth  of  a  cubic  centimeter  of  a  rather  thick 
emulsion  of  carmin  in  salt  solution  (50  centigrams  of  carmin  to  10 
c.c.  of  fluid)  was  slowly  injected  into  the  jugular  vein.  Two  hours 
later  the  leucocyte  count  had  fallen  to  3000.  Another  small 
amount  of  blood  was  taken.  One  cubic  centimeter  of  serum  from 
each  blood  specimen  was  then  placed  in  a  tube  and  inoculated 
with  a  24-hour  culture  of  Massaouah  (1  loop  of  a  culture  suspended 
in  10  c.c.  of  normal  salt  solution). 

NUMBER  OF  COLONIES. 


Time  of  cultures. 

Serum  containing 
11,000  leucocytes. 

Serum  containing 
3,000  leucocytes. 

T 

July  5,  1894,    5  P.M  

9,600 

10,000 

II 

July  5    1894     7  p  M                

0 

2 

ITT 

July  6   1894,  11  A.M.          

0 

Innumerable 

STUDIES  ON   THE   SERUM    OF    VACCINATED   ANIMALS         31 


Frequently,  as  already  mentioned,  the  injection  of  carmin,  even 
though  repeated,  gives  no  evident  hypoleucocytosis.  Under  these 
conditions  there  is  no  difference  in  the  bactericidal  property  of  the 
blood  before  and  after  injection.  Carmin  itself  has  no  effect  on  the 
bactericidal  substances,  as  may  be  shown  by  adding  it  to  serum. 

The  injection  into  normal  animals  of  a  rather  large  quantity  of 
serum  from  an  unvaccinated  animal  brings  about  a  distinct  in- 
crease in  the  bactericidal  property  of  the  serum  of  the  animal.  The 
increased  bactericidal  property  under  these  conditions,  however,  is 
not  to  be  compared  with  that  caused  by  injecting  the  serum  of  vacci- 
nated animals  and,  moreover,  is  not  specific.  The  effect  of  injecting 
normal  serum  is  to  increase  slightly  the  normal  bactericidal  property 
which  is  to  be  found  in  non-immunized  animals,  and  this  property 
is  by  no  means  specific.  We  have  tried  to  determine  whether  the 
bactericidal  property  following  these  injections  is  more  evident  in 
a  fluid  rich  in  leucocytes  than  in  one  containing  only  a  few. 

EXPERIMENT  8.  A  small  amount  of  blood  was  drawn  from  a 
guinea-pig  (leucocytes,  8000).  From  this  blood  was  obtained 
serum  "A";  the  same  guinea-pig  received,  in  the  peritoneal  cavity, 
3  c.c.  of  serum  from  another  normal  guinea-pig.  On  the  following 
day  a  rubber  ring  was  placed  about  the  paw  of  this  animal.  Three 
hours  later  the  edema  was  removed  and  a  small  amount  of  blood 
was  also  taken.  This  blood  contained  6000  leucocytes  and  the 
serum  from  it  was  labelled  serum  "B."  Equal  amounts  of  these 
fluids  were  then  inoculated  with  a  24-hour  culture  of  cholera 
(Oriental  Prussia). 


Time  of  cultures. 

Serum  "A." 

Edema. 

Serum  "B." 

T 

March  7,  1895,    6       P  M  

5,700 

5,400 

6,480 

II. 
TIT 

March  7,  1895,    8       P.M  
March  7    1895    10  30  P  M 

5,520 
6  420 

12,000 
Innumerable 

600 
840 

The  edema  fluid  removed  at  practically  the  same  time  as  the 
blood  that  formed  serum  "B"  may  be  properly  compared  with  this 
serum.  After  4J  hours  at  body  temperature  there  was  no  increase 
in  vibrios  in  serum  "  A";  there  was  a  very  marked  decrease  in  serum 
"B,"  but  in  the  edema  fluid  there  is  an  immediate  and  unrestricted 


32  STUDIES  IN  IMMUNITY. 

increase  of  the  organisms.  Preparations  from  these  fluids  are  even 
more  striking  than  the  plates;  they  show. a  large  number  of  or- 
ganisms in  the  edema  fluid,  very  few  in  serum  "A,"  and  still 
fewer  in  serum  "B."  It  is  also  to  be  noted  that  the  colonies 
grow  more  rapidly  on  the  plates  inoculated  with  edema  fluid  than 
on  the  other  plates. 

The  increase  in  bactericidal  property  in  the  blood  following  the 
injection  of  normal  serum  would  appear  to  be  due  to  a  reaction  on 
the  part  of  the  leucocytes  and  not  to  an  increase  in  the  number  of 
cells  in  the  blood,  as  it  is  not  accompanied  by  a  hyperleucocytosis; 
on  the  contrary,  the  number  of  white  corpuscles  is  found  to  be 
slightly  decreased.  Another  similar  experiment  was  performed 
with  bouillon  in  place  of  normal  serum.  The  same  result  was 
obtained,  and  in  the  same  way  there  was  no  increase  in  leucocytes 
noted  in  the  blood  (8400  before  the  injection  and  7400,  24  hours 
after  injection). 

There  are  certain  undoubted  conclusions  that  may  be  drawn 
from  these  experiments.  //  the  number  of  leucocytes  in  the  blood  is 
diminished  during  life,  the  blood  is  found  to  be  less  bactericidal.  The 
blood  plasma,  more  or  less  completely  separated  from  the  cellular 
elements  within  the  body,  has  less  bactericidal  property  than  the  whole 
blood.  The  separation  of  the  fluid  from  the  cellular  part  of  the 
blood  outside  the  body  by  coagulation  does  not  bring  about  the 
same  result.  The  serum  of  vaccinated  animals  although  deprived 
of  cells  is  energetically  bactericidal.  We  shall  take  occasion  to 
elaborate  these  results  later.  It  seems  reasonable  to  admit  not 
only  that  bactericidal  substances  are  present  in  leucocytes,  but  that 
when  the  blood  has  been  removed  from  the  body  the  white  cor- 
puscles soon  discharge  into  the  serum  those  bactericidal  substances 
which  under  normal  conditions  they  retain  within  themselves.  This 
would  explain  why  the  bactericidal  property  of  body  fluids  which 
is  so  marked  in  vitro  is  much  less  evident  during  life.  This  latter 
fact  has  been  particularly  well  brought  out  by  Metchnikoff,  and 
indeed  it  is  notorious  that  results  obtained  in  vitro  do  not  always 
exactly  indicate  what  goes  on  within  the  animal  body.  Phagocytes 
have  an  efficient  and  rapid  means  of  destroying  the  bacteria  they 
have  taken  hold  of.  The  evidence  of  the  effect  of  phagocytic 
secretions  on  many  organisms,  particularly  on  cholera  vibrios,  is 


STUDIES   ON   THE   SERUM  OF  VACCINATED  ANIMALS        33 

well  shown  in  their  change  in  shape  and  reaction  to  dyes  within  the 
body  of  the  phagocyte.  We  shall  consider  in  the  following  section 
the  nature  of  these  changes  within  the  phagocyte.  There  is  a 
simple  technical  method  that  has  permitted  us  to  study  with 
accuracy  and  facility  a  good  number  of  bacteria  from  this  stand- 
point. 

III.   PHAGOCYTOSIS  IN  VITEO.    THE  BACTERICIDAL  PROPERTY 
OF  PHAGOCYTES. 

Phagocytosis  may  take  place  not  only  in  the  animal  body,  but 
also  in  vitro.  The  phenomenon  of  phagocytosis  occurs  rapidly  in  a 
mixture  of  an  exudate  rich  in  phagocytes  and  a  bacterial  emulsion, 
if  it  be  placed  in  a  moist  chamber  at  a  temperature  of  35  degrees. 

A  good  way  to  obtain  an  exudate  containing  a  large  number  of 
leucocytes  is  to  inject  a  few  cubic  centimeters  of  peptone  bouillon 
into  the  peritoneal  cavity  of  an  animal.  For  example,  if  a  guinea- 
pig  is  given  3  c.c.  of  bouillon  into  the  peritoneal  cavity,  the  exudate 
on  the  following  day  is  found  to  contain  large  numbers  of  white 
corpuscles,  as  Issaeff  *  pointed  out.  In  an  exudate  obtained  in  this 
manner  there  are  found  large  numbers  of  polynuclear  amphophiles, 
considerably  fewer  mononuclear  leucocytes  and  a  very  few  true 
eosinophiles  (that  is  to  say,  leucocytes  with  large  round  eosinophilic 
granules);  these  latter  are  in  such  small  numbers  that  they  are 
frequently  not  to  be  found  on  a  single  slide.  The  exudate  may  be 
drawn  from  the  animal  by  puncturing  the  belly  wall  with  the  point 
of  a  finely  drawn  out  pipette.  As  soon  as  the  peritoneal  cavity  is 
reached  the  exudate  rises  in  the  tube  to  considerable  height. 

A  drop  of  this  exudate  is  placed  on  a  cover  slip.  A  platinum 
loop  full  of  the  culture  fluid,  in  which  the  organism  to  be  studied 
has  grown,  is  then  added  to  this  small  drop  and  mixed  with  it. 
The  cover  slip  is  then  placed  on  a  hollow  ground  slide  and  sealed 
with  vaseline.  After  incubation  for  a  while,  stained  preparations 
are  made  from  this  hanging  drop.  Preparations  stained  with  eosin 
and  then  methylene  blue  (Ehrlich)  are  very  satisfactory.  The  ex- 
udate is  spread  out  on  the  slide  and,  after  drying,  is  fixed  either  by 
heat  (Ehrlich's  method)  or,  better  still,  by  a  5  per  cent  solution  of 
carbolic  acid  or  a  saturated  solution  of  picric  acid.  The  latter 
*  Issaeff,  Zeitschrift  fur  Hygiene,  t.  XVI,  1894. 


34  STUDIES  IN   IMMUNITY. 

method  is  particularly  to  be  recommended  for  preparations  of 
exudates  or  any  body  fluids  containing  albumin.  The  eosin  used 
is  an  alcoholic  solution  (0.5  grams  of  eosin  to  100  c.c.  of  60  per 
cent  alcohol)  and  the  methylene  blue  is  in  saturated  aqueous 
solution. 

In  general,  it  is  best  to  leave  the  inoculated  drop  of  exudate  in  the 
incubator  for  about  4  hours.  By  this  time  the  bacteria  will  not 
have  increased  to  an  unreasonable  extent,  but  phagocytosis  and  the 
changes  caused  in  the  bacteria  by  the  secretion  of  leucocytes  are 
already  quite  manifest.  It  may  be  noted  that  in  a  quarter  of  an 
hour,  at  a  temperature  of  35  degrees,  or  in  even  less  time,  leucocytes 
may  show  a  very  distinct  phagocytic  activity. 

The  exudate  employed  was  obtained  from  guinea-pigs  that 
had  never  received  an  injection  of  bacteria,  but  had  simply  been 
given  bouillon.  A  list  of  the  bacteria  subjected  to  the  action  of 
leucocytes,  with  the  resulting  phenomena  that  we  have  noted, 
follows : 

V.  cholerce  (culture  from  Eastern  Prussia).  —  Polynuclear  leu- 
cocytes have  taken  up  a  large  number  of  the  vibrios.  The  micro- 
organisms outside  the  cells  have  retained  intact  their  form  and  their 
reaction  to  dyes.  Within  the  phagocytes  there  are  vibrios  of 
normal  appearance  colored  a  good  blue,  and  also  numerous  organisms 
that  show  distinct  granulations.  These  granulations  take  different 
shades  of  blue,  light  pink  and  deep  pink.  They  are  quite  like  those 
granules  that  Pfeiffer  noted  in  the  peritoneal  cavity  of  immunized 
animals  after  injecting  a  culture  of  vibrios.  These  oval  or  rounded 
granulations  are  simply  vibrios  that  have  contracted  in  response 
to  the  harmful  secretion  from  the  leucocytes.  The  fact  that  none 
of  these  granulations  are  found  outside  the  cells  shows  very  clearly 
the  superior  bactericidal  power  of  the  protoplasm  of  the  phagocytes 
over  that  of  the  surrounding  fluid.  We  shall  later  consider  these 
granulations  more  carefully  and  also  the  influences  that  produce 
them. 

Some  of  the  vibrios  within  the  leucocytes,  whether  transformed 
into  granules  or  not,  show  marked  changes  in  reaction  to  dyes. 
Instead  of  staining  with  methylene  blue,  the  basic  color,  they  stain 
with  eosin.  Metchnikoff  has  already  observed  vibrios  taken  up  in 
the  animal  body  that  stain  with  eosin;  he  also  noted  the  same  fact 


STUDIES   ON    THE    SERUM   OF  VACCINATED    ANIMALS.        35 

with  the  spirillum  of  recurrent  fever.*  Mesnil  noted  a  similar  fact 
with  the  Bacillus  anthracis.| 

V.  cholerce  (culture  from  Constantinople). —  Same  result. 

B.  typhosus.  —  There  are  many  organisms  within  leucocytes  of 
which  the  majority  have  been  transformed  into  rounded  or  oval 
granules  which  stain  shades  varying  from  blue  to  pink.  The  change 
is  similar  to  that  noted  in  the  cholera  vibrio.  There  are  a  few  intact 
bacteria  within  cells  that  stain  with  eosin.  The  bacilli  that  have 
not  been  taken  up  by  phagocytes  have  a  normal  appearance. 

B.  coli. — The  results  are  similar  to  those  with  B.  typhosus. 
The  same  intracellular  granulation  is  to  be  noted  due  to  the  con- 
traction of  the  organism.  There  are  also  some  bacteria  within  the 
cells  that  appear  swollen.  Stains  are  all  the  way  from  blue  to  red. 
No  change  in  the  organism  outside  the  cells. 

Bacillus  of  Hog  Cholera.  —  The  results  are  also  similar  to  those 
noted  under  cholera  and  typhoid.  Within  the  phagocytes,  in 
addition  to  normal  bacteria,  granules  stained  either  with  blue  or 
with  eosin  are  noted.  The  organisms  outside  leucocytes  are  intact. 

Bacillus  of  Danysz.  —  This  organism  was  discovered  by  Danysz 
in  an  epidemic  among  rats,  and  when  injected  into  these  animals 
causes  a  contagious  and  fatal  enteritis.  The  organism  is  non- 
pathogenic  for  rabbits  and  birds,  and  kills  only  rats,  mice,  and  field- 
mice,  so  that  it  is  actually  being  used  in  agricultural  circles  for  the 
purpose  of  destroying  these  harmful  animals.  It  is  a  cocco-bacillus 
that  does  not  stain  by  Gram  and  resembles  the  colon  bacillus  in 
general.  When  subjected  to  the  leucocytes  of  the  guinea-pig  it  is 
taken  up  with  extraordinary  avidity.  The  phagocytes  take  up  so 
many  bacteria  that  each  one  appears  to  be  stuffed  with  them. 
The  organisms  that  have  been  taken  up,  when  examined  after  the 
usual  period,  are  found  colored  violet  and  at  times  red. 

Bacillus  diphtheria.  —  There  is  a  very  marked  phagocytosis. 
The  shape  of  the  organisms  is  not  changed  (at  least  in  4  hours),  but 
the  reaction  to  dyes  is  extraordinarily  modified.  The  majority  of 
diphtheria  bacilli  taken  up  by  phagocytes  are  colored  a  deep  red  or 
violet.  Outside  the  leucocytes,  on  the  contrary,  the  organism  shows 

*  Cantacuzene,  Mode  de  destruction  du  vibrion  chole>ique  dans  I'organisme, 
1894. 

f  Mesnil,  Sur  le  mode  de  resistance  des  vertebras  inferieurs  aux  invasions 
microbiennes.  Annales  de  1'Institut  Pasteur,  May,  1895. 


36  STUDIES  IN   IMMUNITY. 

its  normal  reaction  for  the  basic  stain.  Mononuclear  leucocytes  also 
have  a  very  distinct  affinity  for  diphtheria  bacilli  and  take  up  a  good 
number  of  them,  but  the  organisms  taken  up  by  the  mononuclears 
show  fewer  color  changes  than  those  taken  up  by  the  polynuclears. 

Bacillus  proteus  vulgaris.  —  The  change  to  a  red  staining  bacillus 
is  still  more  striking  with  Proteus  vulgaris  than  with  the  diphtheria 
bacillus.  After  contact  for  quarter  of  an  hour  with  a  rich  leu- 
cocytic  exudate,  eosin-stained  bacteria  may  be  found  within  the 
phagocytes.  It  is  evident  that  the  protoplasm  of  leucocytes  has  a 
very  marked  action  on  Proteus.  After  a  few  hours'  contact  it  is 
very  hard  to  find  any  organisms  in  the  phagocytes  that  stain  blue. 
But  although  these  bacteria  are  very  much  changed  in  their  chemical 
constitution,  they  show  little  morphological  alteration.  The  bac- 
teria scattered  in  the  surrounding  fluid  are  normal  in  appearance, 
with  the  exception  of  a  few  individuals  that  stain  red.  Proteus  is 
one  of  the  best  organisms  to  demonstrate  phagocytosis  in  vitro, 
and  shows  most  clearly  the  effect  of  leucocytic  fluid. 

B.  anthracis.  —  The  engulfing  of  the  anthrax  bacillus  by  phag- 
ocytes in  vitro  is  easily  demonstrable.  The  polynuclears  take  up 
one  or  two  rods;  when  the  chain  of  bacteria  is  long,  it  frequently 
happens  that  several  phagocytes  join  together  about  the  chain  and 
unite  in  absorbing  it.  The  filaments  that  have  been  taken  up  stain 
red  or  a  shade  between  red  and  blue.  There  are  to  be  noted,  it  is 
true,  in  the  surrounding  fluid  also  bacteria  in  which  changes  are 
quite  evident,  and  it  must  be  admitted  that  the  fluid  of  the  exudate 
seems  to  show  bactericidal  effects  on  this  organism.  But  a  careful 
study  of  smears  shows  clearly  that  these  substances  in  the  surround- 
ing fluid  have  come  by  diffusion  from  the  leucocytes.  It  is  per- 
fectly evident  that  the  red-stained  bacteria  are  more  numerous 
within  the  phagocytes  than  without  them;  and,  what  is  more,  it  is 
not  unusual  to  find  long  threads  of  anthrax  stained  blue  throughout 
their  length,  with  the  exception  of  those  spots  in  contact  with  the 
protoplasm  of  a  leucocyte.  Such  a  picture  shows  well  the  phag- 
ocytic  origin  of  the  soluble  substances  that  are  harmful  for  the 
anthrax  bacillus. 

B.  musco'ides.  —  Abundant  phagocytosis.  Extracellular  organ- 
isms normal.  Within  the  phagocytes  reddish  or  bluish  rods. 

B.  vermicularis.  —  Same  result. 


STUDIES  ON  THE    SERUM  OF    VACCINATED    ANIMALS.        37 

B.  pyocyaneus.  —  Distinct  phagocytosis.  Some  of  the  organisms 
within  the  phagocytes  are  colored  a  faint  pink. 

B.  ramosa  and  the  red  bacillus  of  Kiel.  —  Similar  phenomena. 

Streptococcus.  —  Polynuclear  leucocytes  capture  large  numbers 
of  streptococci.  Within  4  hours  the  majority  of  the  cocci  taken  up 
still  stain  blue.  There  are  to  be  noted,  however,  in  some  phagocytes, 
chains  of  red  streptococci;  at  times  one  or  two  of  the  cocci  in  a 
chain  remain  blue  or  violet. 

It  is  evident  that  the  same  phenomena  may  be  observed  in  vivo 
as  in  vitro.  For  example,  we  have  injected  the  diphtheria  bacillus 
(1  c.c.  of  a  24-hour  bouillon  culture)  into  two  guinea-pigs,  one 
of  which  had  received  the  day  before  3  c.c.  of  bouillon  in  the 
peritoneal  cavity,  and  the  other  of  which  had  received  3  c.c.  of 
anti-diphtheria  serum.  The  injection  of  bouillon  produced  a  rich 
leucocytic  exudate  composed  largely  of  polynuclears,  but  also  con- 
taining a  few  mononuclears.  In  the  animal  injected  with  preventive 
serum,  the  exudate  contained  many  polynuclears,  but,  in  addition, 
a  number  of  mononuclears  (macrophages).  In  both  animals 
phagocytosis  is  noted  after  injection  of  the  culture,  and,  as  is  usual, 
the  polynuclears  seem  to  take  the  greater  part  in  it.  In  the  case 
of  the  guinea-pig  vaccinated  with  serum,  however,  the  macrophages 
are  found  to  have  taken  up  considerable  numbers  of  organisms. 
After  an  hour  there  are  numerous  intraphagocytic  bacilli  staining 
red  both  in  the  animal  that  had  received  bouillon  and  in  the  one 
immunized  with  serum.  Although  the  taking  up  and  destruction 
of  the  organisms  appears  to  go  on  without  any  difficulty,  there 
remain  certain  resistant  bacteria,  so  that  positive  cultures  were 
obtained  from  the  exudates  of  both  animals  18  hours  after  injection. 

The  uniformity  with  which  phagocytosis  occurs  even  when 
leucocytes  have  been  taken  out  of  the  animal  body  and  are  no 
longer  in  their  normal  condition  is  amply  proved  by  these  experi- 
ments.* Changes  of  the  bacteria  in  cells  are  always  noted.  One 

*  The  activity  of  phagocytes  against  bacteria  in  vitro  gives  a  means  of  studying 
the  effect  of  different  poisons,  toxins  or  drugs  on  these  cells.  Active  substances 
may  be  added  to  leucocytes  and  then,  after  varying  periods  of  time,  bacteria  may 
also  be  added.  We  are  at  present  engaged  in  studies  along  this  line.  We  are 
rather  surprised  to  find  that  diphtheria  toxin,  which  kills  the  guinea-pig  in  a  dose 
of  0.2  to  0.1  of  a  cubic  centimeter,  produces  no  effect  on  the  phagocytic  activity 
of  the  leucocytes  of  this  animal. 


38  STUDIES  IN   IMMUNITY. 

of  the  most  interesting  of  these  changes,  as  we  have  already  observed, 
is  the  ease  with  which  certain  bacteria  gain  the  power  of  absorbing 
eosin  instead  of  methylene  blue.  On  account  of  the  regularity  with 
which  this  phenomenon  occurs,  one  is  inclined  to  attribute  the 
pseudo-eosinophilic  granules  in  macrophages  to  bacterial  origin. 
As  for  the  morphological  changes  in  engulfed  bacteria,  it  is  to  be 
noted  that  there  are  present,  in  leucocytes  of  normal  animals  that 
have  received  an  injection  of  cholera  vibrios,  the  same  sort  of 
granules  (transformed  vibrios)  as  in  the  peritoneal  fluid  of  highly 
vaccinated  animals.  In  the  normal  animals,  however,  the  bacte- 
ricidal property,  as  evidenced  by  the  transformation  of  the  vibrio 
into  granules,  is  never  so  energetic,  and  brings  about  the  change 
only  where  the  bactericidal  substance  is  highly  concentrated,  that 
is  to  say,  within  the  phagocytes. 

IV.   LEUCOCYTES  AND  THE  PREVENTIVE  POWER  OF  SERUM. 

It  is  well  known  that  the  blood  of  animals  that  have  been  vacci- 
nated several  times  with  a  culture  of  living  vibrios  killed  by  heat, 
has  not  only  bactericidal  but  preventive  properties.  The  injection 
of  serum  from  such  vaccinated  animals  into  healthy  animals  per- 
mits the  latter  to  withstand  a  dose  of  culture  that,  under  ordinary 
conditions,  is  certainly  fatal.  There  are  preventive  cholera  sera 
which  are  so  very  powerful  that  they  act  in  doses  as  small  as  two 
or  three  milligrams. 

Will  facts  offered  in  explanation  of  the  bactericidal  property  of 
serum  also  explain  the  preventive  power?  Are  the  substances  that 
endow  the  serum  of  vaccinated  animals  with  their  preventive 
properties  present  in  the  body  fluids  of  the  living  animal?  Or  are 
they,  on  the  contrary,  more  or  less  retained  by  the  white  blood 
corpuscles  during  life?  The  experimental  procedures  already 
described  are  also  applicable  to  this  problem. 

It  is  perfectly  evident  that  the  relative  preventive  value  of  serum 
and  of  edema  fluid  from  the  same  vaccinated  animal  may  be  com- 
pared. We  may  also  compare  two  specimens  of  blood  from  the 
same  animal  containing  different  numbers  of  leucocytes,  and  this 
method  we  have  employed.  In  these  experiments  we  used  the 
vibrio  of  Massaouah,  which  is  an  advantageous  culture  for  two 
reasons:  its  virulence  is  relatively  constant,  and,  secondly,  it  is  so 


STUDIES   ON  THE   SERUM   OF  VACCINATED   ANIMALS.       39 

pathogenic  that  there  is  no  necessity  for  injecting  large  amounts  of 
the  culture  into  the  animals,  which  might  be  toxic  apart  from  any 
increase  in  organisms  that  may  take  place.  It  frequently  happens 
in  using  a  culture  of  cholera  vibrios  of  low  pathogenic  power,  but 
of  high  toxicity  (for  example,  the  culture  from  Eastern  Prussia), 
that  well  vaccinated  guinea-pigs  die  on  receiving  a  sub-lethal  dose, 
and  die,  not  of  infection,  but  from  intoxication;  it  is  well  known, 
indeed,  that  protection  by  means  of  serum,  or  active  immunization 
by  means  of  bacteria,  does  not  decrease  the  sensitivity  of  the  animal 
to  the  toxin.  It  is  evident,  then,  that  such  cultures  should  not  be 
used  in  comparing  the  preventive  value  of  two  fluids.  The  Mas- 
saouah  vibrio  employed  kills  guinea-pigs  in  a  dose  of  TV  of  a  24-hour 
agar  culture  intraperitoneally. 

The  preventive  fluids  were  injected  into  the  peritoneum  24  hours 
before  the  intraperitoneal  injection  of  the  culture.  The  agar 
culture  used  for  injection  was  suspended  in  a  definite  amount  of 
salt  solution  (0.6  per  cent)  and  formed  a  homogeneous  emulsion 
that  could  be  easily  measured. 

EXPERIMENT  9.  Rabbit  "A"  had  been  vaccinated  against  the 
Massaouah  culture  and  the  blood  and  edema  fluid  had  been  studied 
in  respect  to  their  bactericidal  properties.  0.25  of  a  cubic  centi- 
meter of  each  liquid  was  injected  into  the  peritoneal  cavity  of  a 
guinea-pig.  The  one  receiving  the  serum  weighed  330  grams,  the 
one  that  received  edema  fluid,  345  grams.  Twenty-fours  hours 
later  these  two  guinea-pigs,  as  well  as  a  control  normal  guinea-pig 
(400  grams),  received  each  TV  of  a  culture.  The  control  died 
rapidly.  The  two  guinea-pigs  that  had  received  protective  injec- 
tions recovered. 

In  this  experiment  both  the  edema  fluid  and  the  serum  were  shown 
to  contain  preventive  properties.  But  it  was  found  by  increasing 
the  amount  of  culture  and  diminishing  slightly  the  amounts  of  the 
preventive  fluids  injected  that  the  properties  are  not  present  in 
equal  amounts  in  the  two  fluids. 

EXPERIMENT  10.  A  guinea-pig  weighing  500  grams  received 
0.2  of  a  cubic  centimeter  of  serum  from  the  vaccinated  rabbit. 
Another  guinea-pig  (525  grams)  received  0.2  of  a  cubic  centimeter 
of  edema  fluid.  A  control  guinea-pig  (535  grams)  received  0.2  of  a 
cubic  centimeter  of  normal  rabbit  serum.  Twenty-five  hours  later 


40  STUDIES  IN  IMMUNITY. 

each  guinea-pig  received  TV  of  a  culture.  The  next  morning  the 
guinea-pigs  that  had  received  the  edema  fluid  and  the  normal  rabbit 
serum  respectively  were  found  dead;  the  guinea-pig  that  had 
received  preventive  serum  was  slightly  sick,  but  soon  recovered. 
There  were  a  number  of  leucocytes  in  the  peritoneal  exudate  of  the 
guinea-pig  that  had  received  the  edema  fluid,  but  very  few  in  the 
control  that  had  received  normal  rabbit  serum. 

Even  when  the  dose  of  culture  used  is  very  large  or  the  guinea- 
pigs  are  very  small,  so  that  the  immunity  conferred  by  the  serum 
is  not  perfect,  there  is  always  to  be  noted  a  distinct  delay  in  the 
death  of  the  animal  receiving  the  preventive  serum  over  the  other 
two.  For  example : 

EXPERIMENT  11.  Guinea-pig  "A"  (245  grams)  received  0.2  of 
a  cubic  centimeter  of  serum;  guinea-pig  UB"  (255  grams),  0.2  of 
a  cubic  centimeter  of  edema  fluid.  Tne  following  day  these  animals 
and  a  control  "C"  (339  grams)  were  given  each  TV  of  a  culture. 
The  following  day  the  guinea-pig  injected  with  edema  and  the  con- 
trol were  found  dead;  the  guinea-pig  injected  with  preventive 
serum  did  not  die  until  the  following  day,  which  is  a  considerable 
delay  when  one  considers  the  usual  rapid  evolution  of  the  perito- 
nitis caused  by  the  cholera  vibrio.  In  such  instances  an  exami- 
nation of  the  peritoneal  exudate  is  interesting.  The  exudate  in  the 
control  is  always  found  to  be  poor  in  leucocytes  and  over-running 
with  vibrios.  The  exudate  from  the  guinea-pig  that  has  received 
edema  fluid  always  contains  considerably  more  cells  than  the  control, 
but  very  much  fewer  than  does  the  exudate  of  the  animal  that  has 
received  serum ;  and  in  this  latter  animal,  too,  the  vibrios  are  very 
much  fewer  in  number.  The  proportion  of  leucocytes  in  the  exudate 
indicates  the  relative  resistance  of  the  individual. 

EXPERIMENT  12.  A  rabbit  had  been  well  vaccinated  against 
the  Massaouah  organism.  Eighteen  days  after  the  last  injection 
an  edema  was  produced.  The  blood  contained  5000  leucocytes 
per  c.m.m. 

(a)  Rather  large  guinea-pigs  were  used.  No.  1  (455  grams) 
received  0.3  of  a  cubic  centimeter  of  edema  fluid ;  No.  2  (430  grams), 
0.3  of  a  cubic  centimeter  of  serum ;  on  the  following  day  these  two 
animals  and  a  control,  No.  3  (450  grams),  received  each  i^  of  a 
culture.  The  control  was  the  only  one  to  die. 


STUDIES   ON  THE   SERUM   OF  VACCINATED  ANIMALS.       41 

(6)  Smaller  guinea-pigs.  No.  1  (240  grams)  received  0.2  of  a 
cubic  centimeter  of  serum;  No.  2  (265  grams),  0.2  cubic  centimeters 
of  edema.  These  two  animals  and  a  control,  No.  3  (300  grams), 
were  given  ro  of  a  culture  on  the  following  day.  The  control  (No. 
3)  and  the  guinea-pig  that  had  received  edema  fluid  were  found 
dead  the  following  day.  The  guinea-pig  that  had  received  serum 
withstood  the  infection. 

(c)  A  guinea-pig  (325  grams)  received  0.2  of  a  cubic  centimeter 
of  serum.  Guinea-pig  No.  2  (350  grams),  0.2  of  a  cubic  centimeter 
of  edema.  Control  guinea-pig  (340  grams).  Each  received  TV  of 
a  culture  the  evening  of  the  same  day.  The  following  morning  the 
control  and  the  animal  that  had  received  edema  fluid  were  found 
dead.  The  guinea-pig  that  had  received  serum  died  two  days  later, 
probably  of  intoxication,  since  the  heart's  blood  was  sterile;  the 
peritoneal  exudate  was  found  to  contain  a  rather  large  number  of 
leucocytes,  which  were  very  infrequent  in  the  exudates  from  the 
other  two  animals. 

Let  us  now  consider  the  preventive  value  of  two  samples  of  serum 
from  the  same  animal,  containing  different  numbers  of  leucocytes. 

EXPERIMENT  13.  A  guinea-pig  that  had  been  well  vaccinated 
against  Massaouah  was  given  an  injection  of  carmin.  A  specimen 
of  blood  was  taken  before  and  after  injection  (serum  "A"  before, 
number  of  leucocytes  11,000;  serum  "B"  after  injection,  number 
of  leucocytes  3000).  The  bactericidal  properties  of  the  two  sera 
were  studied  by  the  method  already  described. 

(a)  Guinea-pig  No.  1  (360  grams)  received  0.3  of  a  cubic  centi- 
meter of  serum  "A."  Guinea-pig  No.  2  (390  grams)  received  0.3 
of  a  cubic  centimeter  of  serum  "B."  Control  guinea-pig  (455 
grams).  The  following  day  each  animal  received  TV  of  a  Massaouah 
culture.  The  control  alone  died. 

(6)  Guinea-pig  No.  1  (345  grams)  received  0.2  of  a  cubic  centi- 
meter of  serum  "A";  guinea-pig  No.  2  (375  grams),  0.2  of  a  cubic 
centimeter  of  serum  "B."  Control  (440  grams).  The  following 
day  each  animal  received  TV  of  a  culture.  The  control  and  the 
animal  that  received  serum  "B"  (containing  the  smaller  number 
of  leucocytes)  were  found  dead  the  following  morning.  The  guinea- 
pig  that  received  serum  "A"  died  the  same  day  in  the  afternoon; 
the  peritoneal  exudate  from  this  animal  was  the  richest  in  leucocytes. 


42  STUDIES  IN  IMMUNITY. 

(c)  Guinea-pig  No.  1  (340  grams)  received  0.2  of  a  cubic  centi- 
meter of  serum  "A";  guinea-pig  No.  2  (350  grams),  0.2  of  a  cubic 
centimeter  of  serum  "B";  control  guinea-pig  (355  grams).  Animals 
given  iV  of  a  culture.  Guinea-pig  No.  2  and  the  control  died  the 
following  afternoon.  Guinea-pig  No.  1,  that  had  received  serum 
"A,"  survived. 

It  is  evident,  then,  that  serum  has  a  higher  preventive  power  than 
plasma,  or,  rather,  than  a  transudate  caused  by  venous  compression 
and  containing  fewer  leucocytes  than  serum.  Serum  containing 
the  normal  number  of  leucocytes  is  more  preventive  than  serum  in 
which  the  leucocytes  have  been  artificially  diminished.  Although 
the  edema  fluid  has,  to  be  sure,  a  distinct  preventive  property,  the 
data  given  indicate  very  clearly  the  importance  of  leucocytes  in  respect 
to  the  preventive  properties  of  serum.  It  is  quite  possible  that  during 
life,  under  normal  conditions,  a  certain  diffusion  of  preventive 
substances  from  the  leucocytes  may  take  place,  although  there  is  no 
certainty  that  the  edema  produced  by  a  rubber  band  corresponds 
at  all  exactly  to  the  plasma  of  the  blood. 

V.  SPECIFICITY  OF  THE  BACTERICIDAL  SUBSTANCE  IN  THE 
SERUM  OF  VACCINATED  ANIMALS. 

In  the  group  of  vibrios  there  are  several  bacterial  species  having 
similar  characters.  It  is  very  difficult  either  by  morphology  or  by 
cultural  characteristics  to  distinguish  the  cholera  vibrio  from  the 
vibrio  of  Massaouah,  the  vibrio  of  Deneke  or  the  vibrio  of  Finkler. 
The  great  importance  of  finding  some  means  of  distinction  between 
these  organisms  accounts  for  the  careful  study  that  has  been  made 
of  them.  On  account  of  its  pathogenic  importance  and  for  the 
value  that  a  positive  diagnosis  of  its  presence  would  have,  the 
cholera  vibrio  has  been  particularly  well  studied  in  order  to  deter- 
mine such  characteristics  as  could  be  used  to  separate  it  from  other 
similar  micro-organisms.  Most  of  the  attempts  at  separation  of 
this  organism  have  failed,  and  it  was  found  that  certain  properties 
which  were  first  supposed  to  be  specific  were  also  present  in  other 
vibrios.  The  comma  bacillus  presents  frequent  confusing  changes; 
it  changes  morphologically;  it  does  not  always  liquefy  gelatin  with 
the  same  rapidity;  and  its  virulence  is  very  changeable  and  easily 
lost.  The  Finkler  organism  or  the  Deneke  organism,  which  are 


STUDIES  ON  THE   SERUM   OF   VACCINATED  ANIMALS.        43 


usually  considered  non-pathogenic,  may,  after  a  few  passages, 
infect  animals  quite  as  well  as  the  typical  cholera  vibrio.  The 
cholera  red  reaction  is  no  certain  method  of  diagnosis.  Morpho- 
logically and  culturally,  then,  the  separation  of  Koch's  bacillus 
from  other  vibrios  is  impossible. 

The  question  arises  as  to  whether  these  vibrios,  which  are  so  much 
alike,  always  react  in  the  same  manner  to  the  bactericidal  sera  of 
vaccinated  animals.  Is  the  bactericidal  property  of  the  serum  of  a 
vaccinated  animal  active  only  against  the  species  of  vibrio  used  to 
vaccinate  the  animal,  or  is  it,  on  the  contrary,  a  more  general  property 
and  active  against  the  majority  of  organisms  belonging  to  the  same 
group?  Pfeiffer  states  that  the  bactericidal  property  in  the  sera 
of  vaccinated  animals  is  specific.  We  have  made  a  few  experiments 
along  this  line.  The  sera  of  several  guinea-pigs  and  rabbits,  each 
of  which  had  been  immunized  against  a  certain  vibrio,  were  tested 
for  their  bactericidal  property  against  several  different  vibrios. 

I.   Serum  of  a  rabbit  immunized  against  the  Massaouah  Vibrio. 

NUMBER  OF  COLONIES. 


I 

J 

1 

a 

E 

s« 

a  "a 

J 

Time  of  cultures. 

S 

o    . 

"8 

0 

2  1 

2 

.2  > 

.2  •§ 

a  o 

|i 

I* 

II 

11 

s  'K 

I.  Apr.  30,  1894,    4.30  P.M. 

16,800 

13,000 

7,800 

13,500 

17,400 

12,000 

II.  Apr.  30,  1894,    6.15P.M. 
III.  May     1,1894,11.15A.M. 

16,200 

8 
0 

0 

0 

70 
120 

16,800 

* 

30 

* 

II.   Serum  of  a  guinea-pig  immunized  against  the  Massaouah 
Vibrio. 


"c 

b 

H 

a 

1 

1 

If 

1 

QO 

Time  of  cultures. 

S 

*8  J3 

fi^ 

""  'i 

«§  -~ 

.2  > 

2    3 

•2  1 

0    § 

.2  g 

a^ 

1  ° 

|a 

P 

|i 

I.  Feb.  20,  1895,  11.30A.M. 

1,150 

1,200 

1,380 

1,280 

600 

II.  Feb.  20,  1895,    1       P.M. 

420 

0 

210 

240 

420 

III.  Feb.  20,  1895,  10       P.M. 

* 

0 

* 

* 

* 

*  Innumerable. 


44 


STUDIES  IN  IMMUNITY. 


III.    Serum  of   a   guinea-pig   immunized    against    the   Vibrio 
Metchnikovi. 


'5 

1 

a 

§ 

« 

i 

=    hi 

E 

S  S- 

Time  of  Cultures. 

S 

0      • 

2^ 

S  ea 

l| 

.2  > 

o-g 

.2  1 

O    3 

o  c 

a-S 

1  o 

i£ 

i« 

I.  March  2,  1895,^2  P.M. 

1,200 

6,600 

12,420 

12,900 

9,000 

II.  March  2,  1895,    5  P.M. 

0 

90 

4,320 

4,800 

5,700 

III.  March  3,  1895,  10  A.M. 

0 

240 

* 

* 

* 

IV.    Serum   of   a   guinea-pig   vaccinated  against   the  Cholera 
Vibrio.     (Eastern  Prussia.) 


I.  Mar.  6,  1895,  4   P.M. 

15,200 

12,400 

16,000 

14,300 

16,800 

II.  Mar.  6,  1895,  6   P.M. 

6,200 

950 

0 

0 

0 

III.  Mar.  7,  1895,  10.30A.M. 

* 

* 

0 

0 

0 

*  Innumerable. 

We  have  not  considered  it  necessary  to  carry  these  experiments 
farther  because  the  results  agree  wholly  with  those  recently  ob- 
tained by  various  observers.  In  fact,  we  have  a  method  which  is 
much  easier  to  employ  than  the  gelatin  plate  method  to  detect 
whether  a  given  vibrio  is  susceptible  to  a  given  immune  serum. 
This  method,  which  we  shall  later  consider,  consists  in  estimating 
the  susceptibility  of  the  vibrio  to  granular  transformation  (Pfeiffer's 
phenomenon)  which  occurs  when  the  organism  is  mixed  either  with 
the  specific  immune  serum  or  the  immune  serum  plus  a  small 
amount  of  fresh  normal  serum.*  It  will  be  seen  in  the  tables  which 
have  been  given  that  the  bactericidal  property  of  a  serum  is  always 
more  active  against  the  vibrio  which  has  served  to  vaccinate  the 
animal  furnishing  the  serum,  or  for  those  few  organisms  that  are 
closely  allied  to  the  immunizing  organism.  There  is  no  doubt  that 
the  bactericidal  effect  is  highly  specialized,  since,  however  similar 
organisms  may  be  morphologically  and  culturally,  certain  of  them 
are  wholly  unaffected  by  a  serum  fatal  to  others.  It  is  to  be  noted 

*  As  will  be  seen  later,  the  immune  serum  must  have  been  recently  obtained  in 
order  to  effect  a  granular  transformation  of  an  organism  without  the  addition 
of  fresh  normal  serum. 


STUDIES   ON  THE   SERUM   OF  VACCINATED   ANIMALS.        45 

that  there  are  several  differences  of  this  sort  in  the  type  organisms 
of  the  species  "cholera  vibrio."  For  example,  the  vibrio  of  Mas- 
saouah  does  not  act  as  do  the  cholera  vibrios  of  Eastern  Prussia, 
Constantinople,  or  Hamburg.  This  does  not  at  all  mean  that  it 
has  not  the  same  pathogenic  properties  as  these  latter.  Immune 
serum  gives  a  delicate  reaction,  but  we  do  not  know  whether  the 
distinctions  it  draws  between  vibrios  are  always  fundamental  ones. 
It  is  certainly  proper  to  collect  all  the  organisms  that  react  in  the 
same  way  to  a  given  serum  into  one  distinct  group  and  to  separate 
this  group  from  all  the  others,  provided  that  the  criterion  of  separa- 
tion is  distinctly  noted.  The  grouping  of  a  given  species  is  not 
always  the  same  if  some  other  criterion  is  used.  There  is  no  means 
of  proving  that  a  classification  based  on  a  reaction  to  serum  is  the 
same  as  a  classification  according  to  pathogenic  power.  There  is 
no  reason  for  saying,  for  example,  that  the  vibrio  of  Massaouah  can- 
not produce  cases  or  epidemics  of  cholera  from  the  simple  fact  that 
it  is  not  affected  by  the  serum  of  animals  immunized  against  the 
cholera  vibrio  from  Eastern  Prussia. 

VI.  THE  NATURE  OF  THE  BACTERICIDAL  SUBSTANCE  IN  VACCI- 
NATED ANIMALS.  ITS  IDENTITY  WITH  THE  BACTERICIDAL 
SUBSTANCES  IN  NORMAL  ANIMALS. 

The  immunized  animal  is  remarkably  well  equipped  for  combating 
the  organism  against  which  it  has  been  vaccinated.  In  its  phag- 
ocytes it  has  a  bactericidal  power  which  would  seem  to  direct  itself 
exclusively  against  that  bacterial  species  against  which  it  has  been 
immunized.  But  even  the  normal  un vaccinated  animal  has  a 
certain  amount  of  protective  power  against  vibrios.  When  vibrios 
are  inoculated  in  normal  serum  there  is  generally  noted  in  the 
beginning  a  decrease  in  their  number.  Although  this  destructive 
power  is  not  distinctly  marked  against  virulent  vibrios,  it  is  more 
marked,  as  Pfeiffer  has  recently  shown,  against  such  vibrios  as  have 
been  grown  for  some  time  on  artificial  media  and  have  therefore 
become  unaccustomed  to  the  body  fluids  and  lost  their  virulence. 
It  is  well  known,  moreover,  that  this  normal  bactericidal  power  may 
be  increased  by  injecting  into  the  animal  a  few  cubic  centimeters 
of  bouillon  or  of  normal  serum. 

There  are  certain  properties  in  common  between  the  bactericidal 


46  STUDIES  IN  IMMUNITY. 

substance  in  normal  animals  and  the  specific  bactericidal  substance 
in  immunized  animals.  They  are  both  destroyed  at  about  60°  C., 
and  sera  heated  to  this  temperature  become  excellent  culture 
media.  In  both  instances  the  power  is  lodged  within  the  phag- 
ocytes. But  these  two  substances  may  be  sharply  distinguished 
from  one  another  by  the  fact  that  the  less  active  substance  of  nor- 
mal serum  is  in  no  sense  specific  and  attacks  vibrios  indifferently, 
while  the  other,  in  vaccinated  animals,  is  very  powerful  and  is 
highly  specific.  The  normal  bactericidal  substance  is  harmful  for 
attenuated  vibrios,  whatever  may  be  their  origin,  but  the  specific 
bactericidal  substance  is  fatal  only  for  a  definite  race  of  vibrios. 
The  two  substances  would  appear,  then,  to  be  quite  different. 

What  is  more,  the  bactericidal  substance  in  the  serum  of  immu- 
nized animals  differs  from  the  preventive  substance  in  the  same 
serum.  The  researches  of  C.  Frankel  and  Sobernheim  *  have  shown 
that  the  preventive  substance  resists  a  prolonged  heating  to  70 
degrees,  whereas  the  bactericidal  substance  is  entirely  destroyed  at 
this  temperature.  Serum  heated  to  70  degrees  is  quite  as  capable  as 
fresh  serum  of  conferring  passive  immunity;  in  other  words,  the  pre- 
ventive substance  is  quite  unaltered  by  this  temperature.  It  may 
be  noted  that  the  essential  property  of  this  preventive  substance 
lies  in  causing  an  intense  bactericidal  property  in  the  treated 
animals,  as  was  well  established  by  Frankel  and  Sobernheim  in 
their  important  study.  These  experimenters  injected  serum  from 
animals  vaccinated  against  cholera  into  guinea-pigs,  and  noted  on 
the  following  day  that  the  serum  of  these  guinea-pigs  had  become 
energetically  bactericidal  for  Koch's  vibrio.  The  same  result  was 
observed  in  animals  given  injections  of  serum  heated  to  70  degrees 
and  thereby  deprived  of  its  own  bactericidal  property.  For  the  mo- 
ment, the  fact  of  importance  to  us  is,  that  the  preventive  substance 
which  immunizes  animals  differs  from  the  bactericidal  substance. 

It  would  seem,  then,  that  we  must  consider  at  least  three  import- 
ant active  substances  in  immunity :  two  different  bactericidal  sub- 
stances, and  a  preventive  substance.  But,  as  a  matter  of  fact,  the 
matter  is  not  so  complicated.  It  would  seem  that  the  bactericidal 
substances  of  normal  and  of  immunized  animals  which  differ  in  the 
matter  of  specificity  are,  in  reality,  quite  identical.  The  feeble  bac- 

*  Fraenkel  and  Sobernheim,  Hygienische  Rundschan,  Nos.  3  et  4,  1894. 


STUDIES  ON  THE  SERUM  OF  VACCINATED  ANIMALS.        47 


tericidal  substance  in  the  normal  serum  acts  energetically  in  the  immune 
serum  owing  to  the  action  of  the  preventive  substance  that  accompanies 
it,  increases  its  power,  and  lends  to  it  its  specific  character. 

As  a  matter  of  fact  it  suffices  to  add  a  small  amount  of  anti- 
cholera  serum,  either  fresh  or  previously  heated  to  60  degrees  and 
so  deprived  of  its  bactericidal  property,  to  normal  serum  to  endow 
the  latter  with  a  very  strong  bactericidal  property.  Two  liquids  then, 
neither  of  which  is  markedly  bactericidal,  form  a  mixture  that  is 
strongly  bactericidal.  As  we  shall  later  see  this  bactericidal  property 
is  specific  and  evidenced  only  toward  that  race  of  bacteria  used  to 
vaccinate  the  animal  from  which  the  preventive  serum  has  been 
obtained.  It  makes  no  difference  in  the  bactericidal  property  if  the 
serum  is  quite  clear  and  without  cells  or  contains  only  a  few  cor- 
puscles. This  fact  may  be  expressed  in  another  way  by  saying  that 
after  the  preventive  serum  has  been  heated  its  intense  and  specific 
property  may  be  restored  by  adding  normal  serum.  This  is  brought 
out  by  the  following  experiment : 

EXPERIMENT  14.  Normal  guinea-pig  serum  recently  obtained 
was  allowed  to  settle  and  the  upper  non-cellular  clear  fluid  was 
taken  off;  the  lower  part  of  the  fluid  contained  red  and  white 
corpuscles.  Tubes  were  made  up  as  follows:  No.  1,  12  drops  of 
serum  from  a  goat  immunized  against  cholera  (Eastern  Prussia) 
(this  serum  had  been  heated  for  one  hour  at  58  degrees).  No.  2, 
12  drops  of  normal  guinea-pig  serum.  No.  3,  8  drops  of  guinea-pig 
serum  plus  4  drops  of  goat  serum,  58  degrees.  No.  4,  identical 
with  No.  3  except  that  the  cellular  part  of  the  normal  guinea-pig 
serum  was  used. 

These  tubes  were  then  inoculated  with  a  24-hour  culture  of  the 
vibrio  suspended  in  10  c.c.  of  normal  salt  solution,  and  gelatin  plates 
were  made  at  intervals. 

NUMBER  OF  COLONIES. 


Time  of  cultures. 

No.  l. 

No.  2. 

No.  3. 

No.  4. 

I. 

April  22,  1895,    6       P.M.  .  . 

8,640 

9,600 

10,200 

9,120 

II. 

April  22,  1895,    7.30  P.M.  .  . 

4,320 

2,160 

0 

0 

III. 

April  22,  1895,  10       P.M.  .  . 

6,480 

3,600 

0 

0 

IV. 

April  23,  1895,  10       A.M.  .  . 

Innumerable 

Innumerable 

0 

0 

48  STUDIES  IN  IMMUNITY. 

It  is  evident  from  this  experiment  that  normal  serum  may  be 
made  strongly  bactericidal  by  the  addition  of  a  small  amount  of 
preventive  serum,  as  shown  by  the  plate  method.  This  fact  may 
also  be  demonstrated  by  adding  a  small  drop  of  a  suspension  of 
vibrios  to  a  drop  of  the  serum  mixture,  when  it  will  be  found  that 
the  organisms  are  rapidly  transformed  into  granules.  Pfeiffer's 
phenomenon,  then,  may  be  produced  in  vitro  as  we  shall  presently 
consider  more  in  detail.  In  the  same  way  that  a  small  amount  of 
preventive  serum  vaccinates  animals  and  gives  them  a  bactericidal 
property  against  the  vibrio,  so  it  would  seem  to  "  vaccinate"  normal 
serum  as  evidenced  by  the  intense  bactericidal  property  which  this 
serum  acquires. 

VII.  THE  SPECIFICITY  OF  THE  PREVENTIVE  SUBSTANCE  IN 
THE  SERUM  OF  IMMUNIZED  ANIMALS. 

The  serum  of  animals  well  immunized  against  a  vibrio  contains 
a  preventive  substance.  This  preventive  substance  is  separate 
from  the  bactericidal  substance  also  present  in  the  serum.  Is  the 
preventive  substance  likewise  specific?  In  other  words  will  the 
serum  of  animals  vaccinated  against  a  vibrio  other  than  Koch's, 
protect  against  infection  with  the  true  cholera  organism?  The 
question  is  still  in  dispute.  Certain  observers  assert  that  the  serum 
of  animals  vaccinated  against  the  vibrio  Massaouah  protects  animals 
from  true  cholera;  but  this  is  denied  by  other  observers.  It  is  easy 
enough  to  perform  experiments  to  elucidate  this  question,  but  the 
interpretations  of  such  experiments  are  confusing.  The  studies  of 
Issaeff  *  have  shown  us  that  the  injection  of  certain  fluids  with  no 
distinct  protective  power  in  animals  may  cause  a  certain  degree  of 
immunity  against  cholera  infection. 

Cholera  peritonitis  develops  rapidly.  Guinea-pigs  that  receive 
injections  of  the  vibrio  are  either  rapidly  killed  or  else  recover 
rapidly,  the  struggle  between  the  animal  and  the  parasite  being 
very  short.  When  a  sub-lethal  dose  is  given  the  rapidity  of  the 
cure  shows  that  the  animal  defense  is  adequate  for  a  time;  it  has 
been  shown,  moreover,  that  vibrios  are  quickly  transformed  within 
the  phagocytes.  On  the  other  hand  the  vibrio  multiplies  and 
secretes  poisons,  and  consequently  if  the  animal  does  not  defend 
*  Issaeff,  Zeitschrift  fur  Hygiene,  XVI,  1894,  283. 


STUDIES  ON  THE   SERUM  OF   VACCINATED   ANIMALS.         49 


itself  immediately  after  inoculation  it  will  soon  find  itself  opposed 
by  a  large  number  of  invaders  and  resistance  will  be  useless.  The 
rapidity  of  defense  then  is  of  considerable  importance  in  the  cure; 
the  slightest  influences  that  hasten  the  reaction  against  an  infection 
may  decide  the  issue  of  the  combat.  Injections  of  bouillon  and  of 
normal  serum  are  preventive  only  by  increasing  slightly  the  bacte- 
ricidal property  and  by  attracting  leucocytes.  These  slight  pre- 
ventive properties  suffice  to  cause  the  animal  defense  to  begin 
immediately  and  so  protect  the  animal.  A  cure  which  in  itself  is 
of  vital  importance  may  be  effected  by  agents  in  themselves  insig- 
nificant. There  is  no  comparison  to  be  drawn  between  the  preven- 
tive power  of  these  inert  fluids  and  that  of  a  specific  serum,  and  it  is 
unsafe  to  judge  of  the  preventive  power  of  a  fluid  simply  by  the 
results  which  it  produces.  For  example,  the  immune  serum  against 
the  vibrio  Metchnikovi  has  an  energetic  attraction  for  guinea-pig 
leucocytes.  If  this  serum  is  injected  into  the  peritoneal  cavity  it 
may  consequently  attract  leucocytes  and  thus  prepare  the  animal 
for  a  defense  against  any  vibrio  that  may  subsequently  be  injected. 
The  most  important  result  caused  by  such  an  injection,  however,  is 
the  genesis  of  an  energetic  bactericidal  power  against  the  vibrio 
Metchnikovi  which  endows  the  leucocytes  with  a  strong  destructive 
power  for  this  vibrio.  But  this  latter  bactericidal  power,  which  is 
so  effectual  against  the  vibrio  Metchnikovi,  is  useless  against  the 
cholera  vibrio. 

EXPERIMENT  15.  A  small  amount  of  blood  was  taken  from  a 
normal  guinea-pig  (serum  I).  The  animal  was  then  given  intraperi- 
toneally  1  c.c.  of  serum  from  a  guinea-pig  vaccinated  against  the 
vibrio  Metchnikovi;  24  hours  later  a  small  amount  of  blood  was 
again  withdrawn  (serum  II).  The  bactericidal  properties  of  these 
two  sera  against  the  vibrio  Metchnikovi  and  against  the  cholera 
vibrio  (Eastern  Prussia)  were  then  determined  by  the  plate  method. 


Serum  I. 

Serum  II. 

Time  of  cultures. 

Vibrio  Metch- 
nikovi. 

Vibrio  chol- 
erae. 

Vibrio  Metch- 
nikovi. 

Vibrio  chol- 
erae. 

I. 
II. 
III. 

March  28,    4       P.M. 
March  28,    7       P.M. 
March  28,  11.30P.M 

4;  500 
12,000 

Innumerable 

2,040 
180 
7,800 

4,800 
0 
0 

1,800 
420 
7,050 

50  STUDIES  IN  IMMUNITY. 

As  is  seen  by  this  table  the  serum  of  the  guinea-pig  before  injec- 
tion of  immune  serum  was  not  strongly  bactericidal  either  for  the 
vibrio  Metchnikovi  or  the  cholera  vibrio.  After  injecting  the  pre- 
ventive serum,  the  serum  of  the  guinea-pig  became  distinctly 
bactericidal  for  the  vibrio  Metchnikovi,  but  not  at  all  so  for  the 
cholera  vibrio. 

The  preventive  value  of  a  given  serum  against  the  vibrio  Metch- 
nikovi and  against  the  cholera  organism  may  be  compared.  The 
animal  treated  with  serum  acquired  a  means  of  defense  solely 
against  the  vibrio  Metchnikovi.  It  is  not  proper  then  to  call  a 
serum  preventive  against  such  and  such  a  vibrio  unless  inoculation 
of  this  serum  into  an  animal  endows  the  latter  with  a  marked  bac- 
tericidal property  for  the  organism  in  question.  The  experiment 
offered  gives  an  example  of  the  specificity  of  the  preventive  power. 
It  by  no  means  proves  that  there  is  no  cholera  vibrio  or  never  will 
be  a  cholera  vibrio  similar  enough  to  the  vibrio  Metchnikovi,  so  that 
an  infection  by  such  an  organism  might  not  be  prevented  by  a 
serum  active  against  the  vibrio  Metchnikovi.  It  indicates  simply 
that  the  preventive  substance  formed  by  the  animal  body  bears  a 
distinct  relation  to  the  bacterium  which  has  formerly  attacked  the 
animal  and  against  which  it  has  become  immunized. 

We  know  that  the  bactericidal  activity  of  immune  serum  is  di- 
rected only  against  that  species  of  vibrio  used  for  immunization.  To 
what  is  this  specificity  in  bactericidal  power  due?  We  have  already 
seen  that  the  bactericidal  substance  in  the  serum  of  immunized 
animals  does  not  differ  from  the  weak  non-specific  substance  in 
normal  animals.  The  specificity  and  strength  of  the  bactericidal 
substance  in  the  serum  of  immunized  animals  must  be  due  to  the 
preventive  substance.  The  specificity  of  the  bactericidal  substance 
indicates  the  specificity  of  the  preventive  substance,  and  the  pre- 
ventive property  is  specific  to  the  same  degree  that  the  bactericidal 
power  is. 

VIII.     SOME  CHARACTERISTICS  OF  THE  PREVENTIVE  SUBSTANCE. 

The  preventive  substances  present  in  the  sera  of  animals  immun- 
ized against  different  organisms  are  not  identical.  The  fact  that  an 
infection  produced  by  one  vibrio  cannot  be  prevented  by  the  serum 
of  an  animal  immunized  against  another  vibrio  is  an  evidence  of 


STUDIES   ON  THE   SERUM  OF  VACCINATED  ANIMALS.        51 

this  difference.  There  is  distinct  correlation  between  the  preventive 
substances  and  the  bactericidal  substances  employed  during  the 
process  of  immunization.  It  seems  hard  to  realize  a  priori  how  an 
animal  can  be  so  delicately  constructed  from  the  chemical  stand- 
point that  according  to  necessity  it  may  build  up  from  its  own 
elements  substances  which  are  preventive,  now  against  one  vibrio 
and  now  against  another.  It  seems,  therefore,  quite  reasonable  that 
the  preventive  substance  may  be  a  simple  bactericidal  substance 
more  or  less  transformed  and  changed.  This  was  the  hypothesis 
that  Buchner  offered  in  such  infections  as  diphtheria  and  tetanus,  on 
the  ground  of  certain  analogies  between  the  properties  of  diphtheria 
toxin  and  antitoxin.  Buchner  thought  that  antitoxin  was  derived 
from  toxin  since  the  preventive  power  of  a  serum  depends  on  the 
amount  of  toxin  that  has  been  injected  rather  than  on  the  real 
resistance  of  the  animal.* 

We  do  not  know  the  nature  of  the  preventive  substance  in  cholera 
serum  and  we  have  no  knowledge  concerning  the  toxin  this 
micro-organism  forms.  We  do  not  know  whether  the  substance 
that  gives  rise  in  the  immunized  animal  to  the  preventive  substance 
is  the  toxin  or  some  other  vaccinating  substance  in  the  culture.  In 
the  extremely  vague  position  in  which  we  are  at  the  present  time  it 
is  best  to  review  the  various  properties  which  the  preventive  sera 
and  the  bacterial  products  are  known  to  possess,  and  to  note  what- 
ever conditions  and  analogies  occur  in  comparing  these  properties. 

The  culture  products  from  the  cholera  vibrio  have  an  attraction 
for  leucocytes.  Does  preventive  serum  have  the  same  property? 
And  does  it  differ  from  normal  serum  in  this  respect? 

We  already  know  that  inoculation  of  preventive  serum  into  the 
peritoneal  cavity  causes  an  increase  in  the  number  of  leucocytes 
there.  It  may  certainly  be  concluded  from  this  fact  that  the  influx 
of  leucocytes  is  due  to  some  attraction  on  the  part  of  the  serum. 
Chemiotaxis,  however,  is  not  the  sole  influence  to  which  the  influx 
of  leucocytes  is  due.  It  is  quite  possible  that  it  may  be  one  of  the 
causes  that  produces  this  collection  of  leucocytes,  but  there  is  cer- 

*  This  fact  has  been  established  not  only  in  tetanus  and  diphtheria,  where  it  is 
a  question  of  toxin  and  antitoxin,  but  also  in  instances  where  this  combination 
does  not  occur,  as,  for  example,  in  the  serum  of  animals  vaccinated  against  the 
hog-cholera  bacillus  studied  by  Metchnikoff. 


52  STUDIES  IN  IMMUNITY. 

tainly  another  factor  that  affects  it,  namely,  the  condition  of  the 
capillary  walls.*  Issaeff  has  already  shown  that  a  substance  with  no 
chemiotactic  influence,  namely,  normal  salt  solution,  when  injected 
into  the  peritoneal  cavity,  increases  the  number  of  cells  there. 

Definite  information  then  may  be  obtained  by  placing  the  sub- 
stance to  be  studied  within  capillary  tubes  closed  at  one  end  and 
leaving  these  tubes  in  the  peritoneal  cavity  for  several  hours. 

If  the  fluid  in  such  tubes  is  a  good  culture  medium,  careful 
asepsis  must  be  practised.  After  the  tubes  are  examined  under 
the  microscope  their  contents  may  be  taken  out  and  spread  on  a 
slide.  With  suitable  stains  the  types  of  leucocytes  present  may  be 
noted  and  also  the  presence  of  any  micro-organism.  The  phenomena 
of  attraction  which  might  be  attributed  to  the  serum  may  be  due 
to  bacterial  secretion,  and  so  it  is  well  when  testing  the  chemiotactic 
property  of  a  given  fluid  to  place  in  the  peritoneal  cavity,  at  the 
same  time,  tubes  containing  a  fluid  of  which  the  chemiotactic 
properties  have  already  been  determined. 

EXPERIMENT  16.  (a)  Four  small  bundles  of  tubes  were  placed 
in  the  peritoneal  cavity  of  a  normal  mouse  at  6  P.M.,  and  withdrawn 
18  hours  later.  Ether  anesthesia  was  administered  during  the 
opening  of  the  peritoneum  and  placing  the  tubes.  The  following 
table  shows  the  effect  of  the  fluids  employed  on  the  leucocytes. 

No.  1.  Twenty-four  hour  agar  culture  of  the  Massaouah  vibrio 
suspended  in  salt  solution  and  sterilized  at  100  degrees.  Strongly 
attracting  for  leucocytes. 

No.  2.  Serum  of  a  rabbit  vaccinated  against  the  vibrio  Mas- 
saouah; this  serum  is  powerfully  preventive  and  bactericidal. 
Distinct  attraction  for  leucocytes. 

No.  3.  The  same  serum  heated  for  an  hour  to  70  degrees  (no 
longer  bactericidal  but  still  preventive).  Distinct  attraction  for 
leucocytes. 

No.  4.   Salt  solution  of  0.65  per  cent.  No  attraction  for  leucocytes. 

In  the  tubes  containing  the  sterile  cholera  culture  the  leucocytes 
form  a  thick  plug  not  extending  very  far  into  the  tube.  In  the  tubes 

*  It  has  already  been  shown  that  leucocytes  approach  certain  substances  owing 
to  a  reaction  to  chemical  substances,  but  it  has  not  been  proved  that  this  chemio- 
tactic influence  is  sufficient  to  affect  leucocytes  through  the  vessel  wall  and  cause 
their  emigration.  It  may  be  that  chemiotaxis  is  of  importance  only  after  the 
leucocytes  have  passed  through  the  wall  of  the  capillary  and  into  the  tissues. 


STUDIES   ON  THE   SERUM  OF   VACCINATED  ANIMALS.        53 

containing  serum  the  white  corpuscles  penetrate  more  deeply  and 
are  more  scattered.  This  difference  in  appearance  is  probably  due 
to  the  fact  that  in  the  serum  the  leucocytes  find  a  suitable  non-toxic 
medium  that  allows  them  to  remain  motile  for  a  longer  period. 

(6)  Tubes  were  placed  in  the  peritoneal  cavity  of  a  normal 
guinea-pig  and  withdrawn  after  12  hours. 

No.  1.  Agar  culture  of  Massaouah  suspended  in  10  c.c.  of  salt 
solution  and  sterilized.  Strong  attraction  for  leucocytes. 

No.  2.  Serum  of  a  rabbit  immunized  against  Massaouah.  Marked 
attraction. 

No.  3.   Serum  of  a  normal  rabbit.     Very  slight  attraction. 

No.  4.   Salt  solution  of  0.65  per  cent.     No  attraction. 

These  experiments  show  that  preventive  serum  has  a  much  more 
marked  chemiotactic  influence  than  normal  serum.  It  may  be 
asked  whether  the  leucocytes  of  a  vaccinated  animal  are  attracted 
by  its  own  serum.  That  such  an  attraction  takes  place  is  shown 
by  the  following  experiment: 

(c)  A  specimen  of  blood  was  taken  from  a  guinea-pig  weighing 
420  grams  that  had  been  well  vaccinated  against  the  vibrio  of 
Massaouah.    Two  days  later  the  serum  was  put  in  tubes  which  were 
placed  in  the  peritoneal  cavity  of  the  same  animal. 

No.  1.  Agar  culture  of  the  vibrio  Massaouah  suspended  in  salt 
solution.  Strong  attraction  for  the  leucocytes. 

No.  2.   Serum  from  a  normal  guinea-pig.     Faint  attraction. 

No.  3.  Serum  from  the  animal  used  in  the  experiment.  Very 
distinct  attraction. 

The  following  experiment  also  shows  that  cultures  of  cholera 
vibrios  will  attract  leucocytes  even  when  they  have  been  suspended 
in  a  rather  large  volume  of  salt  solution. 

(d)  A  normal  guinea-pig  was  used. 

No.  1.  Massaouah  culture  on  agar  24  hours  old  suspended  in  5  c.c. 
of  salt  solution.  Strong  attraction. 

No.  2.  The  same  culture  suspended  in  100  c.c.  of  the  same  solu- 
tion. Strong  attraction. 

No.  3.  Same  culture  suspended  in  200  c.c.  of  the  solution.  Very 
distinct  attraction. 

Considering  these  experiments  collectively  it  is  to  be  noted  that 
the  serum  of  a  normal  rabbit  or  guinea-pig  has  only  slight  attraction 


54  STUDIES  IN  IMMUNITY. 

for  the  leucocytes  of  the  mouse,  of  normal  guinea-pigs,  or  of  guinea- 
pigs  vaccinated  against  cholera.  The  attraction  of  the  serum  of 
immunized  rabbits  or  guinea-pigs  for  leucocytes  is  very  much  more 
marked.  It  will  be  noted  also  that  cultures  of  the  cholera  vibrio 
suspended  in  a  large  amount  of  salt  solution  still  attract  leucocytes 
strongly.  Preventive  serum,  even  when  heated  for  an  hour  to 
70  degrees,  still  attracts  leucocytes. 

The  leucocytes  present  in  largest  numbers  in  these  experiments 
are  the  poly  nuclear  amphophiles. 

Are  there  other  points  of  resemblance  between  the  substances  in 
cultures  and  those  materials  characteristic  of  preventive  serum? 
One  of  the  most  noteworthy  properties  of  this  serum  is  that  it  will 
immunize  with  great  rapidity  even  in  a  small  dose.  It  also  may 
be  shown  that  very  small  amounts  of  a  sterilized  culture  of  cholera 
when  injected  into  the  peritoneal  cavity  24  hours  before  inoculation 
of  the  culture  will  protect  guinea-pigs.  For  example,  1/200  of  a 
twenty-four  hour  agar  culture  of  Massaouah  was  suspended  in  salt 
solution,  sterilized  at  100  degrees,  and  injected.  Controls  that  had 
received  a  corresponding  dose  of  salt  solution  containing  no  bac- 
teria succumbed  to  infection,  whereas  the  guinea-pigs  which  had 
received  the  tiny  doses  of  culture  resisted.  An  even  smaller  dose 
of  an  older  culture  was  equally  successful.  When  mixed  in  equal 
volume  with  a  solution  containing  0.6  per  cent  of  NaCl  and  0.5  per 
cent  of  K2C03  and  then  heated  to  100  degrees  these  small  amounts 
of  culture  still  vaccinate.  In  the  same  way  serum  from  immunized 
rabbits  mixed  in  equal  parts  with  this  solution  and  heated  to  100 
degrees,  does  not  coagulate  and  preserves  for  the  most  part  its 
preventive  properties. 

In  view  of  these  facts  the  hypothesis  that  the  preventive  prop- 
erties of  the  serum  are  due  to  a  conservation  of  certain  vaccinating 
substances  from  the  immunizing  culture  cannot  be  thrown  aside. 
But  if  we  study  the  nature  of  the  immunity  produced  by  small  in- 
jections of  culture  more  attentively  it  appears  to  be  in  no  respects 
similar  to  the  immunity  afforded  by  preventive  serum.  Follow- 
ing the  injection  of  preventive  serum,  the  serum  of  the  animal 
inoculated  acquires  a  strong  bactericidal  power,  but  no  such 
thing  happens  after  the  administration  of  small  amounts  of  killed 
vibrio. 


STUDIES    ON  THE  SERUM  OF  VACCINATED  ANIMALS. 


55 


EXPERIMENT  17.  A  small  amount  of  blood  was  taken  from  a 
normal  guinea-pig  (serum  I).  One  twentieth  of  a  cubic  centimeter 
of  an  old  culture  of  cholera  vibrio  (Eastern  Prussia)  was  then  in- 
jected into  the  peritoneal  cavity.  Twenty-four  hours  later  another 
specimen  of  blood  was  taken  (serum  II) .  The  bactericidal  property 
of  the  two  sera  against  the  vibrio  was  then  determined  by  gelatin 
plates. 

NUMBER  OF  COLONIES. 


Time  of 

cultures. 

Serum  I. 

Serum  II. 

I     March  17    1895 

1  P  M 

3  500 

4  000 

II     March  17,  1895 

,     4  P  M 

420 

510 

III.   March  17,  1895 

,  11  p.M  

Innumerable 

Innumerable 

As  is  seen  from  this  experiment  the  bactericidal  power  is  not 
increased.  It  is  probable  that  the  injection  of  small  amounts  of 
culture  produces  a  relative  immunity  due  to  the  attraction  such 
substances  have  for  leucocytes.  If  preventive  substances  owe  their 
origin  to  bacterial  products,  as  their  specificity  would  indicate,  it 
is  not  owing  to  any  direct  relation  between  the  two,  but  on  account 
of  some  elaboration  on  the  part  of  the  animal  body. 


III.    STUDIES  ON  THE  SERUM  OF  VACCINATED 
ANIMALS* 

BY  DR.   JULES   BORDET. 

PFEIFFER'S   PHENOMENON    (THE    EXTRACELLULAR    GRANULAR 
TRANSFORMATION  OF  VIBRIOS). 

I.   INTERPRETATIONS  GIVEN  TO  PFEIFFER'S  PHENOMENON. 

Pfeiffer  f  found  that  if  a  certain  amount  of  cholera  vibrios  sus- 
pended in  bouillon  is  injected  into  the  peritoneal  cavity  of  a  rabbit  or 
guinea-pig  well  immunized  against  cholera,  that  a  certain  number  of 
these  organisms  undergo  within  a  short  time  an  interesting  change. 
They  lose  their  motility  and  then  contract  into  globules  which  at 
first  are  oval  and  then  rounded.  These  globules  resemble  cocci. 
They  subsequently  become  easily  stained  and  according  to  Pfeiffer 
they  finally  break  up,  and  in  this  way  the  culture  introduced  is 
rapidly  destroyed. 

The  most  important  part  of  Pfeiffer's  discovery  lies  in  the  fact 
that  this  retrograde  transformation  of  the  vibrio  takes  place  for  the 
most  part  outside  the  cells.  As  may  easily  be  imagined  Pfeiffer  has 
drawn  from  these  observations  conclusions  which  would  seem  un- 
favorable to  the  phagocytic  theory.  This  eminent  bacteriologist 
has  still  further  noted  that  the  same  phenomenon  occurs  if  the 
vibrio  is  given  to  a  normal  animal,  provided  a  small  amount  of 
preventive  serum  is  injected  simultaneously.  The  dose  of  this 
serum  varies  naturally  with  its  activity,  but  if  very  strong  the  dose 
is  very  small.  The  serum  causes  the  same  phenomenon  when 
deprived  of  all  bactericidal  property  by  heating  to  60  degrees  or  70 
degrees.  According  to  Pfeiffer  the  phenomena  cannot  occur  with- 
out the  cooperation  of  the  living  animal.  According  to  this  author 

*  See  p.  8. 

t  Pfeiffer,  Zeit.  fur  Hygiene  XVIII,  1894,  1. 
66 


STUDIES  ON  THE  SERUM  OF  VACCINATED   ANIMALS.        57 

the  metamorphosis  of  vibrios  is  quite  apart  from  any  leucocytic  inter- 
vention and  due  to  the  activity  of  the  endothelial  cells  of  the  per- 
itoneum. When  a  normal  guinea-pig  is  given  a  mixture  of  vibrios 
and  preventive  serum  the  endothelial  cells  are  stimulated  and  react 
by  rapidly  secreting  harmful  substances  which  produce  morpholog- 
ical changes  in  the  vibrios  and  then  destroy  them. 

The  transformation  of  the  vibrio,  which  precedes  its  destruction, 
gives  evidence  of  the  harm  that  the  bactericidal  substances  in  the 
animal  have  done.  But  these  substances  as  we  have  already  seen  be- 
long to  the  leucocytes  and  it  is  quite  reasonable  to  imagine,  contrary 
to  the  opinion  of  Pfeiffer,  that  the  bacteria  are  modified  because  in 
the  presence  of  vibrios  the  leucocytes  of  the  peritoneal  cavity  liber- 
ate into  the  surrounding  fluid  the  substances  they  have  formed  and 
which  they  normally  retain.  Leucocytes  indeed  show  the  effects 
of  this  sudden  introduction  of  the  culture  fluid ;  many  cells  become 
motionless  and  swollen  or  broken  up.*  Some  of  them  collect  in 
clumps. 

Metchnikoff  has  just  proved  that  this  latter  reasonable  hypothesis 
represents  the  true  state  of  affairs.  He  was  able  to  produce  Pfeiffer's 
phenomenon  in  vitro  by  adding  to  a  mixture  of  preventive  serum 
and  culture,  leucocytic  extract  from  the  peritoneal  cavity  of  a  nor- 
mal guinea-pig.  This  experiment  evidently  rules  out  the  function 
which  Pfeiffer  has  attributed  to  the  endothelial  cells. 

We  have  pointed  out  in  our  experiments  on  phagocytosis  in 
vitro  |  that  vibrios  that  have  been  taken  up  by  normal  guinea-pig 
phagocytes  may  undergo  a  granular  transformation  identical  with 
the  one  described  by  Pfeiffer.  Since  the  normal  guinea-pig  has 
only  a  faint  bactericidal  power  the  transformation  of  vibrios  goes 
on  only  where  the  destructive  substance  is  most  concentrated,  that 
is  within  the  phagocyte.  But  if  the  preventive  serum  is  added, 
as  Metchnikoff  has  done,  the  bactericidal  power  becomes  much 
more  marked  and  metamorphosis  of  the  organism  may  be  noted 
not  only  in  the  phagocyte  but  also  in  the  surrounding  fluid. 

Whatever  may  be  the  ultimate  conclusions,  Peiffer  has  made  a 
great  contribution  to  the  study  of  immunity.  By  this  visible  and 
microscopically  detectable  alteration  the  vibrio  shows  the  effect 

*  Elie  Metchnikoff,  Annales  de  1'Institut  Pasteur,  June,  1895. 
t  See  p.  33. 


58  STUDIES  IN   IMMUNITY. 

that  body  fluids  have  upon  it  and  acts  therefore  as  an  index  of 
bactericidal  properties  of  these  fluids. 

We  have  done  a  number  of  experiments  with  Pfeiffer's  phenome- 
non for  various  purposes  which  we  shall  consider  collectively  for  the 
purpose  of  exposition.  The  method  employed  was  uniform  and 
may  be  detailed  once  for  all. 

II.  THE  PRODUCTION  OF  PFEIFFER'S  PHENOMENON  IN  VITRO 
THROUGH  THE  COMBINED  ACTION  OF  PREVENTIVE  SERUM 
AND  NORMAL  SERUM. 

Metchnikoff  produced  Pfeiffer's  phenomenon  in  vitro  by  mixing 
the  cholera  vibrio  with  preventive  serum  and  then  adding  leu- 
cocytes drawn  from  the  peritoneal  cavity  to  this  mixture.  These 
leucocytes  may,  however,  come  from  another  source.  We  have 
found  that  the  cholera  vibrio  (Oriental  Prussia)  is  rapidly  changed 
into  rounded  granules  when  placed  in  a  mixture  of  preventive  serum 
and  defibrinated  blood  of  a  normal  guinea-pig.  The  control  of  de- 
fibrinated  blood  without  preventive  serum  gave  a  negative  result. 
When  the  specific  serum  is  absent  the  great  majority  of  the  vibrios 
remain  quite  motile  and  extracellular  changes  only  rarely  occur. 
The  leucocytes  of  the  defibrinated  blood,  to  be  sure,  take  up  a  few 
vibrios  that  become  transformed  within  the  protoplasm  of  the 
leucocyte,  but  the  preventive  serum  is  necessary  to  produce  trans- 
formation in  the  surrounding  fluid. 

Metamorphosis  takes  place  under  these  conditions  quite  as  well  as 
in  the  animal  body,  but  more  rapidly  at  body  temperature  than  at 
room  temperature.  The  defibrinated  blood  used  in  the  experiment 
may  be  obtained  from  various  animals;  guinea-pig,  rat,  rabbit,  or 
goat,  but  human  or  guinea-pig  blood  gives  the  most  distinctive  and 
demonstrable  granulations.  In  preparations  with  rat  or  rabbit 
blood  it  is  rather  difficult  to  find  the  vibrios,  as  the  bactericidal 
effect  is  apparently  so  intense  that  the  granules  rapidly  lose  their 
staining  reaction. 

The  preventive  serum  used  may  have  been  kept  for  some  time, 
but  the  defibrinated  blood  from  the  normal  animal  must  have  been 
obtained  recently.  When  kept  for  a  few  days,  even  if  protected 
from  strong  light,  it  loses  some  of  its  properties. 


STUDIES  ON  THE  SERUM   OF  VACCINATED  ANIMALS.        59 

The  amount  of  preventive  serum  necessary  in  the  experiment  may 
be  very  small.  If  the  serum  is  very  powerful  the  slightest  amount 
brings  about  the  phenomenon  very  well. 

Defibrinated  blood  is  a  mixture  of  serum  and  cells.  Are  the  cells 
necessary  to  produce  Pfeiffer's  phenomenon?  If  we  mix  in  a 
hanging  drop  a  small  amount  of  preventive  serum  and  of  perfectly 
clear  normal  serum,  free  from  cells,  and  then  add  a  suspension  of 
cholera  vibrios,  it  is  found  that  the  organisms  soon  give  a  complete 
transformation  phenomenon.  It  is  evident,  then,  that  the  phe- 
nomenon may  occur  without  the  presence  of  the  cellular  portion 
of  the  blood.  We  have  already  noted*  that  the  addition  of  a 
small  amount  of  preventive  serum,  even  when  heated  to  60  degrees 
or  70  degrees  and  so  deprived  of  bactericidal  power,  endows  normal 
serum  with  strong  bactericidal  property  for  the  vibrio.  We  were 
able  to  demonstrate  this  property  by  the  gelatin  plate  method;  we 
shall  also  show  that  it  may  be  demonstrated  by  the  phenomenon 
of  granular  transformation.  Later  on  we  shall  return  to  a  dis- 
cussion of  the  relation  of  this  experiment  to  immunity.  Let  us 
consider  for  the  present  its  practical  application  as  a  means  of 
diagnosis  of  the  cholera  vibrio. 

III.     DIAGNOSIS  OF  THE  CHOLERA  VIBRIO  BY  PFEIFFER'S 
PHENOMENON  IN  VITRO. 

It  is  well  known  how  difficult  it  is  to  determine  the  exact  proper- 
ties of  the  cholera  vibrio  and  consequently  how  difficult  it  is  to  dis- 
tinguish it  from  other  similar  vibrios.  The  diagnosis  of  the  cholera 
vibrio  in  the  dejecta  of  patients  is  not  of  great  importance  during 
a  declared  epidemic  of  typical  cholera.  It  is  quite  another  matter 
in  isolated  cases  of  choleriform  enteritis.  It  is  very  important 
for  hygienic  reasons  under  these  conditions  to  be  able  to  say  posi- 
tively whether  or  not  the  dangerous  organism  is  present. 

Pfeiffer  and  Issaeff  f  claim  a  specificity  in  the  bactericidal  and 
preventive  power  of  the  serum  of  animals  vaccinated  against 
cholera,  and  the  experiments  that  we  have  mentioned  previously 
led  us  to  corroborate  these  conclusions. 

The  German  scientists  have  used  a  serum  from  animals  immunized 

*  See  p.  47. 

t  Pfeiffer  et  Issaeff,  Deutsche  medicinische  Wochenschrift,  No.  13,  1894. 


60  STUDIES  IN   IMMUNITY. 

against  an  undoubted  cholera  vibrio.  By  injecting  this  serum  they 
produce  a  passive  immunity  in  guinea-pigs  against  the  cholera 
vibrio.  If  a  pathogenic  vibrio  from  a  suspected  case  of  enteritis  is 
subsequently  injected* into  these  immunized  animals  they  do  not 
succumb  if  the  vibrio  is  a  true  cholera  organism.  If,  on  the  other 
hand,  the  animal  does  succumb,  they  conclude  that  the  culture  is 
not  the  authentic  Koch  vibrio. 

Pfeiffer*  has  recently  modified  and  simplified  this  diagnostic 
procedure.  He  inoculates  a  normal  guinea-pig  with  a  bouillon 
culture  of  the  suspected  organism  to  which  a  small  amount  of  anti- 
cholera  serum  has  been  added.  If  the  organism  in  question  is  a 
true  cholera  vibrio,  granular  transformation  takes  place. 

Granular  transformation  is  indicative  of  a  bactericidal  effect  by 
the  surrounding  body  fluid  on  the  vibrio.  This  bactericidal  power 
is  specific  and  affects  only  those  vibrios  which  are  identical  with 
the  one  used  to  immunize  the  animal  from  which  the  serum  is 
taken.  In  addition  to  Pfeiffer,  Dunbar  has  more  recently  shown 
by  numerous  experiments  that  those  organisms  which  resemble 
Koch's  vibrio  culturally  may  be  considered  to  be  true  cholera 
vibrios  if  they  undergo  granular  degeneration  when  injected  with 
cholera  serum  into  the  peritoneal  cavity  of  a  normal  guinea-pig. 
This  means  of  diagnosis  does  not  require  much  serum,  but  is  costly 
on  account  of  the  animals  used  and  the  time  consumed  if  there  are 
a  large  number  of  vibrios  to  examine,  or  if  there  is  no  well-equipped 
laboratory  at  hand.  It  would  therefore  be  very  desirable  to  use 
for  diagnosis  the  method  indicated,  which  consists  essentially  in 
the  production  of  Pfeiffer's  phenomenon  in  vitro  by  means  of  the 
combined  action  of  preventive  serum  and  blood  or  serum  from  a 
normal  animal.  This  method  is  very  easy  and  economical  and 
requires  only  small  amounts  of  preventive  serum  and  fresh  blood. 
A  few  drops  of  blood  obtained  by  puncture  and  taken  in  a  capillary 
tube  will  give  a  drop  or  two  of  serum  which  is  sufficient  to  examine 
several  specimens. 

Let  us  consider  first  whether  the  granular  transformation  in  vitro 
with  our  serum  occurs  with  vibrios  belonging  to  species  other  than 
that  of  the  Koch  vibrio  as  defined  by  Pfeiffer. 

Each  of  the  vibrios  found  in  the  following  table  was  placed  with 
*  Pfeiffer,  Deutsche  medicinische  Wochenschrift,  1894. 


STUDIES  ON   THE   SERUM  OF   VACCINATED  ANIMALS.        61 


a  mixture  of  the  two  sera.     The  sign  4-  indicates  that  a  positive 
granular  transformation  took  place. 


Vibrios. 


Id 

C3  O 

o>  a 


Vibrios. 


Eastern  Prussia •. 

Holland,  A,  B,  C,  D,  E,  F 

Angers 

Dantzig 

Hankin  (Agra) 

Polfer : 

Pilon  (Saint-Denis,  from  Netter) 

Inovrazlaff 

Constantinople,  A,  B,  C,  D,  E,  F, 

G,  H,  I,  J,  K  (Nicolle) 

Cassino. . . 


Hamburg 

Hamburg  (Pfeiffer) 

Paris,  1894 -. 

Saint-Cloud 

Vibrio  Metchnikovi 

Nordhafen 

Finkler 

Olin  (Saint-Denis,  Netter) . 
Sakharoff      )  vibr.  from  Tif 
Rechtsamer  (     Us  water. 
Calcutta. . 


Certain  of  these  vibrios  that  give  Pfeiffer's  phenomenon  will  also 
show  it  to  a  certain  extent  in  normal  serum  without  the  addition  of 
cholera  serum.  These  are  the  slightly  virulent  vibrios  which  are  very 
susceptible  to  the  normal  protective  agents  of  the  animal  body. 
The  most  remarkable  of  these  organisms  in  this  respect  is  vibrio 
"  G."  from  Constantinople.  It  should  be  noted,  however,  that  this 
granular  transformation  of  vibrios  by  normal  serum  is  always  rather 
limited  and  is  rarely  comparable  to  that  caused  by  the  normal  serum 
plus  cholera  serum.  As  a  general  rule  the  addition  of  a  very  small 
amount  of  preventive  serum  suffices  to  produce  the  phenomenon 
in  vitro.  The  technique  that  we  usually  employ  is  the  following: 
A  drop  of  24-hour  culture  suspended  in  6  c.c.  of  salt  solution  is 
placed  on  a  slide;  to  this  drop  is  added  a  loop  of  anticholera  serum. 
To  one  drop  of  this  mixture  is  added  a  similar  drop  of  normal  serum. 
A  slightly  larger  amount  of  serum  is  necessary  to  cause  the  phenom- 
enon in  Pfeiffer's  organism  or  in  the  vibrio  from  Saint-Cloud. 
It  is  not  surprising,  however,  that  certain  varieties  should  resist 
better  than  others. 

Vibrios  that  do  not  give  the  phenomenon  in  vitro  also  fail  to  give 
it  when  injected  with  cholera  serum  into  the  peritoneal  cavity  of 
animals.  There  is  therefore  complete  correspondence  between  these 
in  vitro  experiments  and  experiments  in  the  living  animal.  This 
method,  then,  may  certainly  be  utilized  as  simpler  and  less  expen- 


62  STUDIES  IN  IMMUNITY. 

sive  than  Pfeiffer's  method  of  diagnosis.  Certain  general  rules  of 
technique  should  be  followed  to  insure  uniformity : 

A  24-hour  culture  of  the  vibrio  to  be  examined  is  suspended  in 
from  5  to  7  c.c.  of  bouillon  or  normal  salt  solution.  „  Two  drops  of 
this  suspension  are  then  dropped  from  a  fine  capillary  tube  on  a 
slide  or  in  a  watch  glass;  a  drop  of  preventive  cholera  serum  is  then 
added  (a  serum  supposedly  as  strong  as  our  own) ;  as  a  matter  of 
fact,  this  dose  is  considerably  more  than  is  necessary,  but  it  is  well 
to  use  as  much  as  this  on  the  chance  of  dealing  with  a  very  resistant 
vibrio.  A  small  platinum  loop  of  this  mixture  is  then  taken  and 
placed  on  a  cover  slip,  and  a  small  drop  of  fresh  normal  serum  is 
added  by  means  of  the  same  loop  after  sterilizing  it.  Defibrinated 
blood,  of  course,  may  be  used  in  place  of  serum  or  simply  a  freshly 
drawn  drop  of  human  blood.  The  drop  of  normal  serum  is  mixed 
with  the  other  drop  and  the  cover  slip  applied  to  a  hollow  ground 
slide  and  sealed  with  vaseline.  The  slide  is  then  placed  for  two 
hours  in  the  incubator,  which  time  is  quite  sufficient  to  obtain  a 
complete  transformation.  We  emphasize  these  details  because  the 
three  factors  — vibrio,  normal  serum  and  preventive  serum  —  should 
be  used  in  rather  exact  proportions.  It  is  well  to  make  two  control 
preparations  at  the  same  time  and  by  the  same  method.  One  of 
these  should  contain  a  typical  Koch  vibrio  that  is  known  to  give  the 
phenomenon  rapidly  with  active  serum.  From  this  control  we 
learn  whether  enough  of  each  serum  has  been  used.  A  second  con- 
trol is  made  with  the  vibrio  to  be  examined,  normal  serum,  and 
a  drop  of  salt  solution  in  the  place  of  the  preventive  serum.  This 
second  control  will  indicate  whether  the  organism  in  question  is 
very  attenuated  and  so  liable  to  transformation  by  the  normal 
serum  alone  without  any  cholera  serum.  If  the  second  control  also 
gives  Pfeiffer's  phenomenon,  which  is  unlikely,  no  definite  conclu- 
sion may  be  drawn  as  to  the  pathogenic  nature  of  the  organism 
examined. 

Since  the  preparations  remain  only  two  hours  at  body  tempera- 
ture, perfect  asepsis  to  prevent  growth  of  other  contaminating 
bacteria  is  unnecessary.  Examination  may  be  made  either  in  the 
fresh  condition  or  by  means  of  stained  preparations.  There  is  fre- 
quently some  difficulty  in  staining  the  granules,  particularly  when 
the  preparation  has  been  left  too  long  in  the  incubator  and  the 


STUDIES   ON  THE  SERUM  OF  VACCINATED   ANIMALS.        63 

reaction  has  gone  too  far.  Carbolated  thionin  is  a  good  stain  to  use. 
The  slide  is  fixed  by  heat  and  stained  for  one  minute  with  thionin 
and  then  differentiated  in  water  until' there  is  no  longer  any  notice- 
able diffusion  of  the  stain  (about  ten  minutes  when  there  are  many 
blood  corpuscles  present  or  four  or  five  minutes  when  clear  serum 
has  been  used).  The  granules  stain  a  purplish  blue;  the  red  blood 
corpuscles  greenish.  If  the  preparations  are  left  too  long  in  water, 
the  granulations  will  be  completely  decolorized.  The  formation  of 
thionin  crystals  may  be  avoided  by  drying  the  preparation  with 
filter  paper.  The  preparations  may  also  be  stained  with  a  con- 
centrated aqueous  solution  of  methylene  blue  or  by  a  very  dilute 
solution  of  Ziehl's  fuchsin.  When  the  methylene-blue  stain  is  used, 
fixation  by  a  saturated  solution  of  picric  acid  is  recommended,  with 
a  counterstain  of  alcoholic  eosin. 

This  method  is  evidently  only  a  modification  or  rather  a  simplifica- 
tion of  the  method  that  Pfeiffer  has  described.  It  depends  on  the 
same  principle  and  its  value  is  equally  open  to  criticism.  Are  we 
indeed  sure  of  the  diagnostic  value  of  these  results?  Owing  to  the 
specificity  of  bactericidal  sera,  it  seems  to  us  that  if  an  organism 
agrees  culturally  and  gives  this  phenomenon  with  a  true  anticholera 
serum  we  may  identify  it  as  a  true  cholera  vibrio.  But  if,  on 
the  other  hand,  we  are  dealing  with  an  organism  obtained  from  a  case 
that  clinically  resembles  cholera,  and  this  organism  possesses  the 
morphological  and  cultural  peculiarities  of  Koch's  vibrio  but  fails 
to  give  this  reaction  with  cholera  serum,  we  should  not  be  author- 
ized in  saying,  with  our  present  knowledge,  that  the  vibrio  in  ques- 
tion is  not  identical  with  the  cholera  organism  and  that  the  case 
is  not  one  of  true  cholera.  Different  cholera  vibrios  from  various 
sources  show  variations  in  their  resistance  to  serum.  More  serum 
is  necessary,  for  example,  to  transform  the  Saint-Cloud  vibrio  than 
the  vibrio  from  Eastern  Prussia.  Although  the  sera  that  one 
employs  are  active,  they  have  nevertheless  their  limitations.  If 
the  Saint-Cloud  organism  had  been  slightly  more  resistant  or  the 
serum  we  used  slightly  weaker,  we  should  not  have  obtained  the 
phenomenon.  It  is  far  from  being  proved  that  there  may  not  be 
organisms,  which,  although  essentially  true  cholera  organisms  and 
of  the  same  spe'cies  as  the  Koch  organism,  may  not  have  been 
more  highly  differentiated  as  regards  resistance  to  destructive 


64  STUDIES  IN  IMMUNITY. 

organic  substances  than  usual.  And,  what  is  more,  it  has  by  no 
means  been  proved  that  organisms  that  differ  slightly  from  Koch's 
organisms  are  incapable  of  producing  cholera.  For  example, 
the  vibrio  of  Massaouah,  which  is  unlike  the  cholera  organisms 
usually  met  with,  is  unquestionably  a  cause  of  cholera.  (Fermi's 
experiment.)  This  organism,  however,  does  not  fulfill  Pfeiffer's 
condition. 

Nevertheless,  since  the  majority  of  cholera  vibrios  recovered 
from  the  stools  of  cholera  cases  give  the  phenomenon,  the  method 
may  be  recommended,  with  a  certain  reserve  as  to  negative  results. 


IV.    PFEIFFER'S  PHENOMENON  WITH  THE  VIBRIO 
METCHNIKOVI. 

Will  all  vibrios,  even  the  most  virulent  of  them,  undergo  trans- 
formation outside  the  protoplasm  of  leucocytes  when  injected  into 
the  peritoneal  cavity  of  specifically  immunized  guinea-pigs?  Is 
the  vibrio  Metchnikovi,  the  virulence  of  which  is  recognized, 
easily  altered  by  the  peritoneal  fluid? 

We  injected  ^  of  a  24-hour  agar  culture  of  vibrio  Metchnikovi 
into  a  guinea-pig  immunized  by  repeated  injections  of  killed  or 
living  cultures.  The  serum  of  this  animal  was  highly  preventive. 
The  organisms  were  rapidly  clumped,  but  very  few  of  them  were 
morphologically  changed.  After  an  hour,  for  example,  in  addition 
to  the  majority  of  intact  vibrios,  a  few  individuals  were  found  in 
these  clumps  broken  up  into  fine  granulations  and  others  that 
stained  poorly,  but  were  morphologically  intact.  The  typical 
transformation  into  large  granulations  was  only  to  be  found  in 
the  protoplasm  of  polynuclear  leucocytes  which  contained  intact 
vibrios  as  well.  One  or  two  hours  after  injection  the  leucocytes 
were  found  clumped  together.  Later  they  increased  in  number  and 
many  were  seen  scattered  throughout  the  fluid;  6  hours  after 
injection  the  number  of  leucocytes  was  relatively  large  and  the 
vibrios  were  few.  In  a  preparation  from  the  exudate  6  hours 
and  20  minutes  after  injection  an  unphagocyted  and  morpho- 
logically intact  clump  of  vibrios  was,  however,  noted.  It  is 
evident,  then,  that  not  all  vibrios  undergo  granular  transformation 
equally  well. 


STUDIES   ON  THE   SERUM   OF  VACCINATED    ANIMALS.        65 

V.   FACTORS  CONCERNED  IN  THE  PRODUCTION  OF  PFEIFFER'S 
PHENOMENON  IN  VITRO. 

Let  us  consider  the  experiment  for  producing  the  granular 
transformation  of  the  cholera  vibrio  in  vitro  by  the  combination 
of  normal  serum  and  serum  of  an  immunized  goat.  We  repeat 
that  it  is  not  necessary  for  cells  to  be  present  either  in  the  preven- 
tive serum  or  in  the  normal  serum  to  produce  a  complete  Pfeiffer's 
phenomenon. 

There  are  three  factors  necessary  in  the  experiment ;  each  of  these 
factors  may  be  studied  in  turn  and  we  may  determine  whether  there 
are  conditions  that  render  these  factors  unfit  to  produce  the  phe- 
nomenon and  also  whether  any  one  of  them  may  be  omitted  or 
replaced  by  any  other  factor.  The  first  factor  is  the  cholera  vibrio. 
We  have  already  seen  that  the  cholera  vibrio  from  Eastern  Prussia 
which  was  used  in  producing  the  anticholera  serum  may  be  replaced 
by  any  other  of  the  true  vibrios.  There  are  two  other  factors:  the 
normal  serum  and  the  preventive  serum.  Is  the  presence  of  both 
of  these  substances  necessary?  Or  may  one  of  them  be  omitted  and 
the  phenomenon  still  occur?  These  are  the  first  questions  that  arise. 
We  already  know  that  the  normal  serum  employed  has  little  or  no 
power  in  itself  to  change  the  form  of  the  vibrios. 

The  preventive  serum  used  in  these  studies  was  from  a  goat  that 
had  been  well  vaccinated  against  the  Eastern  Prussia  vibrio.  It 
produces  no  transformation  of  vibrios  in  any  dose  when  not  asso- 
ciated with  normal  serum.  When  used  alone  the  only  effect  that 
this  serum  has  on  vibrios  is  to  immobilize  them.  A  very  small 
amount  of  serum  rapidly  paralyzes  their  motility.  It  is  also  to  be 
noted  that  the  vibrios  collect  in  small  clumps  in  the  fluid.* 

The  serum  we  first  used  had  been  drawn  from  the  goat  three  weeks 
previous  to  our  experiments.  It  might  reasonably  be  supposed 
that  during  this  period  the  serum  had  lost  some  of  its  properties, 
since  it  is  known  that  the  bactericidal  substance  is  easily  affected. 

*  We  have  not  been  able  to  decide  whether  this  clumping  is  an  active  phenome- 
non due  to  the  vibrio  itself  or  a  purely  physical  affair.  If  the  red  blood  cells 
of  a  normal  guinea-pig  are  placed  in  their  proper  serum,  they  diffuse  uniformly  and 
soon  collect  at  the  bottom  of  the  hanging  drop.  But  when  our  protected  goat 
serum  is  added  to  such  normal  serum  the  corpuscles  collect  in  small  separate 
clumps  and  give  the  drop  a  granular  appearance. 


66  STUDIES  IN  IMMUNITY. 

And  this  idea  proved  to  be  correct.  We  then  tried  recently 
obtained  serum  from  the  same  animal.  This  fresh  serum  brings 
about  transformation  of  the  vibrio  without  the  addition  of  normal  serum. 
The  phenomenon,  to  be  sure,  was  not  complete;  there  were  a  few 
vibrios  that  remained  unaltered  and  others  that  seem  to  have  been 
killed  without  any  modification,  since  they  failed  to  take  the  stain. 
The  immunized  goat  serum  is  not  as  suitable  a  medium  in  which  to 
demonstrate  transformation  as  is  the  serum  or  blood  of  a  vaccinated 
guinea-pig.  Fresh  immune  serum  from  this  latter  animal  also  brings 
about  complete  transformation  without  the  slightest  necessity  for  the 
addition  of  normal  serum. 

Let  us  consider,  first,  a  preventive  serum  which  does  not  alone 
cause  the  granular  transformation.  Since  the  aid  of  normal  serum 
is  necessary,  we  may  better  study  the  influences  that  affect  its 
activity. 

All  the  following  experiments  have  been  modeled  after  a  funda- 
mental experiment  and,  in  order  to  be  comparable  with  it,  they 
have  all  been  performed  in  an  identical  manner;  a  single  factor,  the 
normal  serum,  has  been  modified.  The  hanging  drops  examined 
were  always  made  in  the  same  way: 

The  hanging  drops  were  made  from  two  equal-sized  drops  placed 
side  by  side  on  the  slide  and  then  mixed.  These  two  drops  were 
each  from  the  same  platinum  loop.  One  of  the  drops  consists  of 
the  fluid  whose  bactericidal  property  is  tested  (in  this  case  the  nor- 
mal serum)  and  the  other  consists  of  an  emulsion  of  vibrios  (24-hour 
agar  culture  suspended  in  6  c.c.  of  normal  salt  solution)  mixed  or 
not,  as  the  case  may  be,  with  the  preventive  serum.  This  latter 
mixture  was  made  as  follows:  A  drop  of  the  emulsion  of  vibrios 
was  dropped  from  a  capillary  tube  into  a  watch  glass  and  to  it  a 
large  platinum  loop  of  preventive  serum  was  added.  The  pre- 
ventive serum,  then,  in  the  final  hanging  drop  is  very  small  in 
amount,  much  less  than  the  normal  serum.  Controls  were  also 
made  in  each  instance  by  using  an  emulsion  to  which  no  preventive 
serum  had  been  added.  An  experiment  may  be  represented  by 
this  abbreviation: 

Cholera  +  preventive  goat  serum  4-  normal  serum, 
or 

Cholera  +  normal  serum. 


STUDIES   ON  THE  SERUM  OF  VACCINATED  ANIMALS.        67 

In  the  tables  that  follow  the  +  sign  indicates  that  the  granular 
transformation  took  place.  Now  that  we  have  considered  the 
technique  certain  points  may  be  considered. 

First.  Does  the  method  of  observation  indicated  show  delicate 
differences  in  bactericidal  power  between  two  fluids? 

It  is  already  known  that  normal  serum  alone  will  sometimes  pro- 
duce a  partial  transformation  of  the  vibrio,  but  this  effect  is  relatively 
slight.  We  also  know  that  the  injection  of  normal  serum  or  even 
bouillon  into  the  peritoneal  cavity  of  a  normal  guinea-pig  increases 
within  certain  limits  the  non-specific  bactericidal  property  of  this 
guinea-pig's  serum.  This  increase  in  bactericidal  property  may  be 
detected  by  our  method.  A  small  amount  of  blood  is  taken  from 
a  guinea-pig;  the  animal  is  then  given  4  c.c.  of  normal  guinea-pig 
serum  and  24  hours  later  a  new  specimen  of  blood  is  taken.  The 
effect  of  these  two  sera  on  the  cholera  vibrio  without  the  addition 
of  cholera  serum  is  then  determined.  A  relatively  weak  culture  is 
used.  Since  no  preventive  serum  is  used  the  transformation  of  the 
vibrios  with  either  of  these  sera  is  only  partial.  It  may,  however, 
be  noted  that  the  serum  obtained  from  the  animal  after  injection 
of  normal  serum  causes  metamorphosis  in  many  more  vibrios  than 
does  the  serum  obtained  before  injection;  there  are  also  many  more 
non-motile  vibrios.  The  difference  between  the  two  preparations 
is  very  distinct  and  demonstrates  the  delicacy  of  this  method.  We 
repeat  that  the  phenomena,  although  relatively  different  in  degree, 
are  only  partial  in  both  instances  and  incomplete  unless  a  drop  of 
preventive  serum  is  added. 

Second.  Is  the  bactericidal  substance  of  normal  serum  still  present 
in  the  blood  of  animals  that  have  succumbed  to  certain  infections  or 
intoxications?  Is  it  also  present  in  the  animals  that  have  been  immu- 
nized against  organisms  other  than  the  cholera  vibrio? 

The  blood  of  a  guinea-pig  that  had  died  of  anthrax,  the  blood  of 
a  rabbit  that  had  just  succumbed  to  a  pneumococcus  infection,  the 
pleural  exudate  of  a  guinea-pig  that  had  died  of  diphtheria  toxin, 
and  the  blood  of  a  guinea-pig  that  had  been  vaccinated  against 
cholera  and  had  died  of  cholera  toxin,  all  gave  Pfeiffer's  phenomenon 
in  the  presence  of  cholera  serum.  The  blood  of  the  last  animal,  in 
spite  of  the  fatal  intoxication,  brought  about  Pfeiffer's  transfor- 
mation without  the  addition  of  cholera  serum,  that  is,  the  blood 


68  STUDIES  IN   IMMUNITY. 

remained  both  bactericidal  and  preventive.  The  persistence  of  the 
preventive  power  under  these  conditions  has  already  been  noted  by 
Metchnikoff.  The  blood  of  guinea-pigs  immunized  against  the 
vibrio  Metchnikovi  or  the  Massaouah  vibrio  and  incapable  of 
producing  the  phenomenon  alone,  produces  it  very  well  when  added 
to  the  preventive  cholera  serum. 

The  bactericidal  substance,  then,  is  to  be  found  generally  in  ex- 
perimental animals  and  persists  even  after  fatal  infections.  It 
occurs  both  in  the  blood  of  vaccinated  animals  and  in  normal 
animals. 

Third.  The  effect  of  heat  on  the  fresh  defibrinated  blood  (or  serum) 
of  a  normal  guinea-pig. 

Fresh  blood  serum  from  a  normal  guinea-pig  was  placed  in 
several  tubes.  One  of  these  tubes  was  left  unheated ;  the  others 
were  heated  for  5  minutes  to  temperatures  of  50  degrees,  55  degrees, 
60  degrees,  and  64  degrees  respectively. 


Hanging  drops.  Phen°m- 


Cholera  +  preventive  goat  serum  4-  unheated  normal  serum  . . 


+  normal  serum  50° 
+  normal  serum  55° 
+  normal  serum  60° 
+  normal  serum  64° 


enon 


From  this  table  we  may  conclude  that  the  bactericidal  substance 
of  normal  serum  necessary  to  produce  Pfeiffer's  phenomenon  is 
destroyed  if  the  serum  is  heated  for  5  minutes  to  from  50  degrees 
to  55  degrees.  It  may  be  noted  that,  even  in  those  tubes  where 
there  was  no  transformation,  the  vibrios  were  motionless  and 
clumped  owing  to  the  continued  presence  of  the  preventive 
serum. 

Fourth.  The  effect  of  heat  on  the  preventive  serum.  Pfeiffer  has 
recently  shown  that  preventive  serum  heated  to  65  degrees  still 
produces  metamorphosis  of  the  vibrio  when  placed  in  the  peritoneal 
cavity  with  the  cholera  organism.  The  same  fact  is  true  in  vitro. 
It  is  interesting  to  consider  the  effect  of  heat  on  quite  fresh  serum 
from  an  immunized  guinea-pig;  as  we  have  already  seen,  this  serum 


STUDIES  ON  THE  SERUM  OF  VACCINATED   ANIMALS.         69 

alone  produces  Pfeiffer's  phenomenon  without  the  aid  of  normal 
serum.  Tubes  of  fresh  serum  from  well-immunized  guinea-pigs 
were  heated  to  50  degrees,  55  degrees,  60  degrees,  and  64  degrees 
for  5  minutes.  Each  one  of  these  sera  was  then  mixed  with  an 
emulsion  of  cholera. 


Hanging  drops.  Phenom- 

enon 


Cholera  (Eastern  Prussia)  +  normal  G .  p .  serum 

+  unheated  preventive  serum  +  normal  G.  p.  serum 


-f  unheated  preventive  serum. 
+  preventive  serum  50 
+  preventive  serum  55 
+  preventive  serum  60 


preventive  serum  60°  +  normal  G.  p.  serum 


As  may  be  seen  from  this  table,  serum  heated  to  50  degrees  trans- 
forms the  vibrio;  heated  above  this  temperature  it  has  no  effect. 
The  bactericidal  substance  in  the  serum  of  immunized  animals, 
then,  is  destroyed  at  the  same  temperature  as  in  normal  animals. 
But  this  heated  serum,  which  alone  cannot  produce  Pfeiffer's  phe- 
nomenon, brings  it  about  perfectly  well,  and  apparently  with  undi- 
minished  vigor,  when  normal  serum  is  added.  In  other  words, 
normal  serum  restores  to  the  immune  serum  the  property  lost  by 
heat.  A  mixture  of  two  fluids/each  of  which  alone  has  no  bacteri- 
cidal property,  forms  a  fluid  that  has  high  bactericidal  property  for 
a  specific  organism.  We  have  already  described  this  experiment 
and  in  the  previous  description  we  demonstrated  the  regeneration 
of  bactericidal  power,  not  by  means  of  Pfeiffer's  phenomenon,  but 
by  gelatin  plate  cultures. 

What  conclusions  may  be  drawn  from  these  facts?  We  have 
already  considered  them  partially  and  these  experiments  only 
confirm  them.  It  would  seem  as  if  the  serum  of  vaccinated  animals 
had  no  particular  bactericidal  substance,  but  that  a  similar  bactericidal 
substance  is  present  in  the  blood  of  normal  as  well  as  of  immunized 
animals.  This  bactericidal  substance  is  not  specific  unless  mixed 
with  the  preventive  substance,  and  under  its  normal  conditions  will 
affect  only  attentuated  vibrios.  Its  energetic  action  depends  on 
the  combined  presence  of  a  preventive  substance  that  is  present  only  in 


70  STUDIES  IN   IMMUNITY. 

the  serum  of  immunized  animals.  It  may  be  that  this  specific  pre- 
ventive substance  has  of  itself  some  harmful  effect  on  the  vibrios 
that  predisposes  them  to  feel  the  power  of  the  bactericidal  sub- 
stance. At  least  the  preventive  substance  alone  has  no  real  anti- 
septic properties,  for  it  cannot  kill  a  culture  or  even  prevent  its 
growth.  It  lends  to  the  bactericidal  substance  present  with  it, 
however,  a  character  of  specificity.  When  fresh,  the  serum  of 
vaccinated  animals  has  both  substances,  but  when  heated  to  55  degrees 
for  a  few  moments  or  kept  for  a  long  time,  the  preventive  substance 
alone  remains;  the  addition  of  fresh  serum  is  necessary  to  restore 
to  it  its  bactericidal  property.  It  is  indifferent  whether  this  fresh 
serum  containing  the  bactericidal  substance  comes  from  a  normal 
animal  or  an  immunized  animal,  or  even  from  an  animal  that  has 
just  died  of  such  an  infection  as  that  caused  by  anthrax  or  the 
pneumococcus. 

Fifth.  Is  the  bactericidal  substance  present  in  equal  amounts  in 
normal  and  in  immunized  animals  ?  While  the  immunized  animal 
is  forming  the  specific  preventive  substance,  does  it  increase  to  any 
extent  the  bactericidal  substance  present  before  immunization? 
One  may  determine  at  least  in  an  approximate  manner  the  amount 
of  bactericidal  substance  present  in  normal  and  immune  serum. 
Let  us  mix  a  loop  of  well-immunized  guinea-pig  serum  with  a  drop 
of  culture.  We  have  established,  let  us  suppose,  by  a  preliminary 
experiment,  that  a  drop  of  this  mixture  placed  with  a  drop  of  normal 
serum  gives  a  complete  Pfeiffer's  phenomenon;  that  is  to  say,  in  a 
drop  of  this  emulsion  there  is  enough  preventive  substance  present. 
It  must  be  noted  that  the  fresh  preventive  substance  alone  may 
give  metamorphosis  of  the  vibrio.  But  under  the  conditions  noted, 
that  is,  one  loop  of  the  serum  to  one  drop  of  cholera  emulsion, 
no  distinct  phenomenon  takes  place.  But,  as  we  have  already 
shown,  there  is  enough  preventive  substance  there ;  it  is  the  bacteri- 
cidal substance  that  is  lacking.  If  we  add  another  loop  of  the  same 
serum  to  the  mixture,  the  phenomenon  occurs  very  distinctly  and 
becomes  still  more  complete  if  a  third  loop  is  added.  At  the  same 
time,  if  we  add  normal  serum  to  a  similar  mixture  of  cholera  emul- 
sion and  active  preventive  goat  serum,  we  find  that  only  three 
loops  are  necessary  to  bring  about  the  usual  degree  of  the  phe- 
nomenon. The  amount  of  fresh  serum,  whether  from  a  normal  or 


STUDIES   ON  THE  SERUM  OF  VACCINATED   ANIMALS.        71 

from  an  immunized  animal,  necessary  to  add  to  the  preventive 
substance  is  in  either  case  little  and,  within  broad  limits,  apparently 
the  same.  It  seems  evident,  then,  that  the  bactericidal  substance 
in  immune  serum  is  not  sensibly  greater  in  amount  than  in  normal 
serum. 

VI.   LEUCOCYTIC  SECRETIONS  AND  PFEIFFER'S  PHENOMENON. 

Experiments  that  have  been  given  in  a  previous  article*  showed 
us  that  during  life  the  bactericidal  substance  is  within  the  leucocytes. 
When  blood  is  taken  out  of  the  vessels  this  substance  is  liberated  in 
the  surrounding  medium  and  endows  the  serum  with  bactericidal 
properties.  Both  serum  deprived  of  some  of  its  leucocytes,  and 
edema  fluid,  are  less  bactericidal  than  serum  obtained  under  normal 
conditions.  They  are  also  less  preventive.  It  is  easy  to  determine 
whether  edema  will  produce  Pfeiffer's  phenomenon  when  added  to 
preventive  serum.  Hanging  drops  are  prepared  containing  on  the 
one  hand,  cholera  vibrio,  preventive  serum  and  serum  of  a  nor- 
mal guinea-pig;  and,  on  the  other  hand,  cholera  preventive  serum 
and  edema  fluid  from  the  same  normal  guinea-pig.  These  mixtures 
are  prepared  in  the  usual  manner,  so  that  the  doses  are  equal  in  each 
preparation. 


Hanging  drops. 

Phenom- 
enon 

Cholera  (Eastern  Prussia)                +  normal  G.  p.  serum  

o 

+  normal  G.  p.  edema  

o 

+  preventive  goat  serum  -4-  normal  G.  p.  serum 

_j_ 

+  preventive  goat  serum  +  normal  G  •  p«  edema  .               „ 

o 

It  is  to  be  noted  that  edema  fluid  causes  no  Pfeiffer's  phenomenon. 
It  is  evident  that  the  bactericidal  substance  in  this  fluid  is  too 
small  in  amount,  since  there  is  complete  transformation  in  the  prep- 
aration containing  serum.  In  the  same  manner  it  may  be  deter- 
mined that  goat  milk,  aqueous  humor  from  the  guinea-pig,  and 
urine,  tears,  or  saliva  (human)  when  added  to  preventive  serum 
produce  no  metamorphosis  of  vibrios.  Edema  fluid  from  a  guinea- 
pig  infected  with  anthrax,  and  pleural  exudate  from  a  guinea-pig 

*  See  page  24  et  seq. 


72  STUDIES  IN  IMMUNITY. 

killed  by  diphtheria  toxin,  both  produce  the  phenomenon;  in  these 
latter  fluids  numbers  of  leucocytes  are  present. 


Hanging  drops. 


Cholera  (Eastern  Prussia)  -f  normal  G.  p.  serum. 

+  preventive  goat  serum  +  normal  G.  p.  serum. 


preventive  goat  serum  +  normal  goat  milk 


4-  preventive  goat  serum  +  human  urine. 

+  preventive  goat  serum  +  human  tears 

+  preventive  goat  serum  -f  human  saliva 

+  preventive  goat  serum  +  G.  p.  aqueous  humor. 

+  preventive  goat  serum  +  edema  anthrax  G.  p. 


preventive  goat  serum  +  pleural  ex.  dipt.  G.  p.  . 


What  is  the  action  of  edema  fluid  from  a  guinea-pig  that  has  been 
highly  immunized  against  cholera?  It  is  known  that  fresh  serum 
from  this  animal  will  alone  produce  the  phenomenon,  since  it  con- 
tains both  the  bactericidal  and  the  preventive  substances.  It  is 
therefore  of  interest  to  determine  whether  the  edema  fluid  from 
such  a  guinea-pig  will  lack  one  or  both  of  these  substances.  Edema 
fluid  alone,  without  either  normal  serum  or  preventive  serum,  does 
not  cause  the  phenomenon.  But  are  both  substances  or  is  only 
one  of  them  lacking?  To  determine  whether  the  bactericidal 
substance  is  lacking,  a  hanging  drop  is  prepared  containing  the 
vibrio,  preventive  serum  and  edema  fluid.  Controls  are  made  as 
indicated. 


Hangin,  drops. 


Cholera  (Eastern  Prussia)  +  normal  G.  p.  serum 

-f  preventive  goat  serum  +  normal  G.  p.  serum 

+  preventive  goat  serum  +  vaccinated  G.  p.  edema.  . . . 


From  this  table  it  is  evident  that  the  edema  fluid  from  immunized 
animals  as  well  as  that  from  normal  animals  contains  no  demon- 
strable bactericidal  substance.  But  is  the  preventive  substance 
entirely  lacking?  It  will  be  recalled  that  in  previous  experiments 
the  edema  fluid  was  shown  to  be  distinctly  preventive  for  animals 
although  inferior  in  this  respect  to  serum.  In  the  following  table 


STUDIES   ON   THE  SERUM   OF  VACCINATED   ANIMALS.        73 

preparations  were  made  with  vibrio,  edema  fluid  and  normal  serum, 
and  transformation  takes  place: 


Hanging  drops. 

Phenom- 
enon. 

Cholera 

(Eastern  Prussia) 

+ 

normal 

G. 

P 

serum  

0 

+  preventive 

goat 

serum    + 

normal 

Q. 

P 

serum  

+ 

+  vaccinated 

G 

•P- 

edema  + 

normal 

G. 

P- 

serum  

+ 

It  is  evident,  then,  that  the  edema  fluid  contains  enough  of  the 
preventive  substance,  of  which  a  very  small  amount  is  necessary 
to  produce  the  phenomenon,  and  it  may  be  added  that  it  is  difficult 
to  obtain  an  edema  fluid  without  leucocytes.  Even  if  there  were 
no  leucocytes  present  and  it  were  distinctly  proved  that  leucocytes 
are  necessary  for  the  presence  of  the  preventive  substance,  it  might 
well  be  that  a  certain  amount  of  it  would  be  liberated  during  life 
into  the  fluids  of  the  body  and  eventually  be  excreted;  it  is  well 
known,  indeed,  that  the  preventive  power  falls  rapidly  when  injec- 
tions are  stopped.  But  there  is  a  body  fluid  that  under  normal 
conditions  is  almost  entirely  deprived  of  cells,  that  is,  the  aqueous 
humor.  It  may  be  shown  that  the  aqueous  humor  in  vaccinated 
guinea-pigs  has  neither  bactericidal  nor  preventive  power.  Whether 
mixed  with  normal  serum  or  with  immunized  goat  serum,  aqueous 
humor  produces  no  change  in  the  vibrio. 


Hanging  drops. 

Phenom- 
enon 

Cholera  (Eastern  Prussia)  +  normal  G.  p.  serum 

+  preventive  goat  serum  +  normal  G.  p.  serum. 

+  vaccinated  G.  p.  aq.  humor  +  normal  G.  p.  serum 


+  preventive  goat  serum  +  vaccinated  G.  p.  aq.  humor 


It  would  seem,  therefore,  that  the  function  of  leucocytes  in  the 
genesis  of  the  specific  properties  of  serum  is  well  determined. 

In  a  consideration  of  bactericidal  and  preventive  power  we  have 
covered  several  of  the  more  important  body  fluids.  We  may  now 
consider  intestinal  transudate  from  the  same  point  of  view.  In 
human  cholera  the  vibrio  is  not  a  parasite  in  the  tissues;  it  invades 


74  STUDIES  IN   IMMUNITY. 

the  organism  only  after  death  and  during  life  usually  remains  in  the 
intestine;  that  is  to  say  its  culture  medium  is  the  intestinal  secretion. 
It  is  important,  therefore,  to  consider  the  properties  of  this  secretion. 
Metchnikoff,  as  we  know,  succeeded  in  producing  true  intestinal 
cholera  in  young  rodents.  His  method  consists  in  introducing  the 
cholera  vibrio  accompanied  by  certain  bacteria  that  facilitate  their 
development,  into  the  stomach  of  these  animals.  Under  these 
conditions  cholera  is  regularly  produced.  When  one  autopsies 
young  rabbits  treated  in  this  way  the  intestine  is  found  filled  with 
a  rather  clear  fluid  swarming  with  vibrios.  Metchnikoff  has  further 
determined  that  this  intestinal  cholera  may  be  produced  in  animals 
that  have  received  large  injections  of  a  strong  preventive  serum 
quite  as  well  as  in  animals  which  have  had  no  previous  treatment. 
In  such  animals  the  vibrio  increases  in  the  intestine,  and  remains 
motile  and  unmodified.  It  may  be  easily  shown  that  intestinal 
secretion  plus  our  preventive  serum  causes  no  metamorphosis  of 
the  vibrio.  The  same  negative  result  is  obtained  if  normal  serum 
be  added  to  the  intestinal  fluid  from  a  young  rabbit  killed  by  cholera. 
This  animal,  however,  had  received  a  previous  dose  of  cholera  serum 
and  its  blood  was  strongly  bactericidal  for  the  vibrio.  An  intes- 
tinal secretion  of  this  sort  contains  no  leucocytes ;  it  does  contain, 
however,  epithelial  cells  which  apparently  give  the  fluid  no  distinct 
activity. 

Does  the  cholera  vibrio  become  accustomed  to  bactericidal  sera? 
Can  we  by  repeated  passage  through  bactericidal  fluids  adapt  it  so 
that  it  no  longer  feels  the  bactericidal  effect  with  the  same  intensity 
and  fails  to  give  granular  transformation? 

If  we  inoculate  a  rather  large  quantity  of  the  cholera  vibrio  (East- 
ern Prussia)  in  a  mixture  of  fresh  guinea-pig  serum  and  immunized 
goat  serum  the  metamorphosis  takes  place  rapidly.  But  if  the 
amount  inoculated  is  sufficient  the  vibrio  will  eventually  grow  and 
after  24  hours  a  considerable  number  of  normal  organisms  will  be 
found. 

A  few  drops  of  this  growth  may  then  be  inoculated  in  a  mixture 
similar  to  the  first  and  when  this  culture  has  grown  out  a  new 
transfer  made.  If  this  transfer  is  repeated  twenty  times  we  find 
that  after  each  inoculation  a  rapid  metamorphosis  of  the  vibrio 
occurs,  and  even  after  two  days,  when  the  culture  has  finally  grown 


STUDIES  ON  THE  SERUM  OF  VACCINATED   ANIMALS.         75 

out,  a  great  number  of  granules  will  be  found  with  the  normal 
vibrios. 

After  twenty  passages  through  bactericidal  serum,  the  vibrio 
is  quite  as  apt  to  be  transformed  as  in  the  beginning.  It  is  quite 
evident,  then,  that  the  vibrio  acquires  no  tolerance  and  becomes  no 
less  susceptible  to  the  bactericidal  effect  of  the  fluid.  It  continues 
to  show  a  granular  change  which  is  not  strictly  a  disintegration, 
but  an  active  contraction  of  the  organism  which  would  appear  to 
serve  some  purpose.  In  this  new  form  it  presents  in  a  given  volume 
a  smaller  surface  of  contact  to  the  surrounding  fluid,  and,  therefore, 
better  avoids  its  harmful  effect. 

The  fact  that  the  vibrio  does  not  become  accustomed  to  contact 
with  the  bactericidal  substances  from  leucocytes  explains  the  diffi- 
culty in  increasing  its  virulence.  After  a  few  passages  through  the 
animal  body  a  definite  virulence  is  reached  which  is  not  easily  in- 
creased. The  cholera  vibrio  does  not  seem  to  possess  the  characters 
necessary  for  a  dangerous  general  parasite  and  does  not  give  gener- 
alized infections.  It  is  not  necessary  for  the  organism  to  acquire 
a  great  resistance  to  the  bactericidal  properties  of  the  animal  body 
in  order  to  live  in  the  intestinal  contents  and  form  toxins  there, 
for,  as  we  have  already  shown,  no  bactericidal  substance  is  present 
in  the  intestinal  secretion.  It  is,  therefore,  by  no  means  proved  that 
a  vibrio  that  is  only  slightly  pathogenic  in  the  peritoneal  cavity 
may  not  be  very  pathogenic  when  present  in  the  digestive  tract. 

REMARKS  ON  THE  MECHANISM  OF  IMMUNITY. 

I.    ACTIVE  IMMUNITY.    BACTERICIDAL  PROPERTIES  OF  LEUCOCYTES.     THE 
CONCEPTION  OF  A  "  UNITY  OF  THE  BACTERICIDAL  SUBSTANCE." 

Active  immunity  is  the  immunity  shown  by  animals  vaccinated 
by  repeated  injections  of  living  or  sterilized  cultures  of  bacteria  or 
of  bacterial  products.  It  is  established  slowly,  but  it  is  much  more 
enduring  than  immunity  brought  about  by  preventive  sera.  This 
form  of  immunity  is  accompanied  by  cellular  changes  that  are 
sufficiently  lasting  to  create  temporarily  in  the  immunized  animals 
properties  that  are  not  to  be  found  in  normal  animals. 

The  study  of  immunity  has  convinced  us  that  the  resistance  of 
animals  to  pathogenic  organisms  is  regularly  due  to  the  activity  of 
phagocytes.  In  a  previous  article  we  have  considered  the  more 


76  STUDIES  IN   IMMUNITY. 

important  proofs  of  the  accuracy  of  this  conception  and  the  facts 
brought  out  in  this  article  do  not  change  our  opinion.  It  has  been 
established  that  in  vaccinated  animals  the  phagocytes  have  their 
protecting  properties  distinctly  increased.  Not  only  is  the  tactile 
sensitivity  of  their  phagocytes  more  perfect  as  Massart  has  shown,* 
so  that  they  can  take  up  the  struggle  with  their  adversaries  more 
preparedly,  but  we  believe  that  it  may  be  granted  that  the  bac- 
tericidal properties  of  their  body  fluids  are  increased.  In  certain 
cases,  as  with  the  vibrios,  the  antiseptic  property  of  leucocytes 
is  increased  to  such  an  extent  that  it  is  liberated  in  the  surround- 
ing fluid,  either  on  removing  the  blood  from  the  body  or  by  some 
harmful  effect  on  the  leucocytes.  The  substances  diffused  from 
these  leucocytes  in  the  blood  fluid  may  then  bring  about  destruc- 
tive changes  in  bacteria.  When  serum  is  endowed  with  bactericidal 
properties  it  owes  them  to  the  leucocytes;  the  bactericidal  power  of 
serum  is  only  a  reflection  of  the  power  within  leucocytes,  and  is 
present  only  in  a  relatively  feeble  degree. 

A  bactericidal  property  is  not  always  present  in  the  serum  of 
immunized  animals.  The  sera  from  animals  vaccinated  against 
tetanus,  diphtheria,  hog-cholera,  etc.,  do  not  destroy  their  respective 
organisms.  May  we  conclude  in  these  instances  that  the  bacterici- 
dal substance  is  lacking  in  the  serum?  Must  we  make  a  distinction 
between  those  instances  in  which  the  serum  of  immunized  animals 
is  bactericidal  and  those  in  which  it  is  not?  Since  the  bactericidal 
power  of  serum  indicates  the  destructive  power  of  phagocytes,  are 
we  authorized  in  these  instances  of  diphtheria  or  hog-cholera  in 
considering  the  absence  of  bactericidal  properties  in  serum  as  indic- 
ative of  little  bactericidal  activity  in  the  phagocytes? 

Such  a  conclusion  would  not  be  legitimate.  It  would  seem  rather 
that  there  is  a  regular  increase  of  bactericidal  property  in  phagocytes 
in  immunity  and  that  we  know  now  only  of  certain  general  examples 
of  this  fact.  And  if  the  increase  of  this  property  is  not  evident  in 
certain  cases  by  a  notable  increase  in  the  bactericidal  property  of 
the  serum,  it  is  apparently  due  to  the  fact  that  certain  micro- 
organisms possess  a  relatively  higher  resistance  to  such  properties. 
Not  all  bacteria  are  equally  easily  destroyed  by  those  diluted  bac- 

*  J.  Massart,  Le  chemiotaxisme  des  leucocytes  et  I'lmmunite*.  Annales  de 
1'Institut  Pasteur,  May,  1892. 


STUDIES  ON   THE   SERUM  OF   VACCINATED   ANIMALS.        77 

tericidal  substances,  which,  although  strong  within  the  leucocytes, 
are  necessarily  weakened  when  liberated  into  the  serum.  When 
phagocytosis  of  the  cholera  vibrio  by  the  leucocytes  of  a  well- 
immunized  animal  is  studied  in  vitro  the  granular  transformation 
of  the  organism  and  its  changes  in  color  reaction  are  found,  not  only 
in  the  leucocytes,  but  also  in  the  surrounding  fluid.  When  such  an 
experiment  is  made  with  the  diphtheria  bacillus  and  the  leucocytes 
of  a  guinea-pig  immunized  the  day  before  by  a  large  dose  of  anti- 
diphtheria  serum  (3  c.c.),  it  is  found  that  the  organisms  within  the 
leucocytes  lose  their  power  to  stain  blue  and  take  eosin,  whereas 
outside  the  leucocytes  the  bacteria  show  no  tinctorial  change.  In 
other  words,  the  activity  has  remained  within  the  protoplasm  of  the 
phagocyte.  It  is  well  known,  moreover,  as  Behring  has  shown  us, 
that  the  body  fluids  of  animals  immunized  against  diphtheria  have 
no  effect  on  Loffler's  bacillus,  which  differs  from  the  findings  in  the 
case  of  cholera.  And,  although  there  is  apparently  a  different  set 
of  facts  to  consider  in  each  of  these  cases,  it  is  to  be  noted  that  ener- 
getic bactericidal  activity  on  the  part  of  the  phagocytes  occurs  in 
both  instances. 

If  there  is  a  distinction,  then,  to  be  drawn  between  bactericidal 
sera  and  those  that  are  not,  it  is  due  to  a  difference  in  resistance  of 
the  specific  organisms  and  not  to  the  absence  or  presence  of  a 
bactericidal  substance  in  the  serum. 

The  best  evidence  of  this  fact  is  that  the  more  strongly  animals 
are  immunized  the  more  frequently  do  we  find  examples  of  the 
humoral  bactericidal  activity  and  extracellular  destruction  of 
bacteria.  In  other  words,  the  destructive  function  has  been 
increased  to  such  a  point  that  it  is  manifest  even  outside  the 
phagocytes.  The  use  of  highly  immunized  animals  was  necessary 
to  discover  Pfeiffer's  phenomenon.  Later  Pfeiffer  *  and  Dunbar  f 
found  that  the  typhoid  bacillus  also  shows  granular  transformation 
when  injected  into  the  peritoneal  cavity  of  an  animal  together  with 
a  certain  amount  of  preventive  serum.  Dunbar  has  noted  similar 
facts  with  phosphorescent  vibrios  and  Bacillus  pyocyaneus.  These 
observations  agree  entirely  with  those  originally  made  on  the 

*  Pfeiffer,  Deutsche  medicinische  Wochenschrift,  No.  48,  1894. 
t  Dunbar,  Zum  Stande  des  bakteriologischen  Cholera  Diagnose.      Deutsche 
medicinische  Wochenschrift,  February,  1895. 


78  STUDIES  IN  IMMUNITY. 

cholera  vibrio  by  Pfeiffer.  The  analogy  is  so  complete  that  it 
seems  justifiable  to  assert  that  the  substance  that  gives  rise  to  the 
morphological  change  in  the  colon  bacillus,  the  typhoid  bacillus, 
and  the  phosphorescent  vibrios  is  of  leucocytic  origin  as  well  as  in 
the  case  of  the  cholera  vibrio.  Indeed,  when  B.  typhosus  or 
B.  coli  is  mixed  with  leucocytes  from  a  normal  guinea-pig  the 
transformation  occurs  only  inside  cells.  Oval  granulations  are 
formed  there  similar  to  the  cholera  granulations.  It  is  evident, 
then,  that  Pfeiffer's  phenomenon,  whether  with  the  colon  bacillus  or 
the  typhoid  bacillus  or  Koch's  vibrio,  follows  the  same  rule.  The 
colon  bacillus  and  the  typhoid  bacillus  give  further  examples  of 
the  fact  that  phagocytes  may  become  so  active  in  well-immunized 
animals  as  to  bring  about  a  diffusion  of  their  substances  into  the 
surrounding  fluid. 

We  have  already  seen  that  the  bactericidal  substance  that  affects 
vibrios  is  not  in  itself  specific.  It  is  the  preventive  substance  that, 
while  adding  to  its  activity,  endows  it  with  specificity.  We  have 
shown,  accordingly,  that  bactericidal  sera  from  animals  immunized 
against  various  organisms  are  to  be  distinguished  only  by  the 
specificity  of  their  preventive  substance.  The  bactericidal  sub- 
stance, properly  speaking,  in  immunized  animals  is,  with  the  possible 
exception  of  certain  quantitative  changes,  quite  the  same  as  in 
normal  animals.  And  since  Pfeiffer's  phenomenon  occurs  with  bac- 
teria other  than  the  cholera  vibrio  and  under  identical  conditions, 
it  seems  evident  that  the  bactericidal  substance  that  phagocytes  use  in 
their  struggle  against  bacteria  is  the  same,  whatever  may  be  the  organism 
attacked.  In  an  animal  immunized  against  a  given  infection  the 
bactericidal  substance  is  directed  against  the  specific  organism  on 
account  of  the  presence  of  the  preventive  substance,  the  specificity  of 
which  depends  on  the  organism  used  for  immunization.  It  is  due  to 
the  presence  of  this  peculiar  preventive  substance  that  the  animal 
directs  its  destructive  power  against  a  given  organism.  What  is 
the  nature  of  this  active  and  distinctive  body?  By  what  mechanisn 
have  the  body  cells  been  enabled  to  create  this  specific  substance 
that  is  so  valuable  to  the  animal?  This  is  one  of  the  most  important 
problems  of  cellular  biology,  and,  at  the  same  time,  at  present  the 
greatest  enigma  with  which  immunity  confronts  us. 


STUDIES   ON  THE  SERUM   OF  VACCINATED   ANIMALS         79 


II.  THE  MECHANISM  OF  THE  IMMUNITY  CONFERRED-  BY  PREVENTIVE 
SERUM  AND  THE  EVIDENCE  FOR  CONSIDERING  THIS  IMMUNITY  AS  A 
PURELY  CHEMICAL  PHENOMENON. 

There  are  certain  conclusions  to  be  drawn  from  the  facts  that  we 
have  offered  as  to  the  nature  of  the  immunity  conferred  by  serum. 

Cholera  serum,  as  we  know,  is  not  antitoxic;  animals  vaccinated 
against  cholera  have  no  real  immunity  against  cholera  toxin.  The 
serum  gives  an  immunity  only  against  the  organism  itself.  It 
contains,  when  fresh,  two  substances:  a  bactericidal  substance  and 
a  preventive  substance.  Serum  that  has  been  kept  for  some  time 
or  heated  to  55  degrees,  no  longer  contains  the  bactericidal  sub- 
stance. As  Fraenkel  and  Sobernheim  were  the  first  to  show,  such 
a  serum,  although  deprived  of  its  bactericidal  substance,  still 
retains  its  immunizing  properties. 

It  is  easy  to  understand  why  the  presence  of,  this  bactericidal 
substance  is  not  indispensable  for  the  preventive  action  of  the  serum. 
It  is  due  to  the  fact  that  the  bactericidal  substance  is  not  a  peculiar 
property  of  this  serum  but  exists  in  any  normal  serum,  so  that  a 
normal  animal  given  an  injection  of  cholera  serum  is  protected 
because  it  already  possesses  the  bactericidal  substance. 

The  substance  that  the  normal  animal  does  not  have  is  the  pre- 
ventive substance,  and  it  is,  therefore,  this  latter  substance  that  it 
is  important  to  furnish.  One  may  ask  how  this  substance  when 
introduced  into  the  tissues  produces  immunity.  It  is  certain  that 
leucocytes,  in  common  with  other  sensitive  living  cells,  react  to  the 
presence  of  the  preventive  serum.  Stimulated  by  this  serum  they 
give  evidence  of  positive  chemiotaxis.  We  know,  moreover,  that 
in  animals  immunized  by  serum,  phagocytosis  occurs  with  remark- 
able intensity.  It  is  difficult,  then,  in  view  of  these  facts,  not  to 
believe  in  the  idea  advanced  by  several  observers  and  notably  by 
Metchnikoff,  Roux,  and  Sanarelli  that  serum  acts  on  cells  as  a 
"stimulin"  that  excites  phagocytosis. 

But  there  is  another  phenomenon  in  passive  immunity  which  is 
perhaps  even  more  important.  The  injection  of  preventive  cholera 
serum  causes  the  appearance  of  a  very  distinct  bactericidal  property 
against  the  cholera  vibrio,  for  which  the  serum  injected  is  specific. 
We  know  already  that  the  preventive  substance,  incapable  in  itself 


80  STUDIES  IN   IMMUNITY. 

of  destroying  the  vibrio,  when  mixed  with  normal  serum,  which  in 
itself  is  only  faintly  bactericidal,  acquires  new  and  energetic  destruc- 
tive properties.  It  is  not  necessary  to  imagine  any  reaction  on  the 
part  of  cells,  due  to  preventive  serum,  to  form  this  normal  substance 
contributed  by  the  normal  serum,  since  it  has  been  shown  to  be 
regularly  present  in  the  normal  animal.  This  substance  is  not 
uniformly  dissolved  in  the  plasma  during  life,  but  is  confined  rather 
within  the  leucocytes. 

What  happens,  then,  when  preventive  serum  is  inoculated?  The 
active  substances  penetrate  into  the  leucocytes  as  we  already  know 
by  the  fact  that  leucocytes  show  evidence  of  chemiotaxis  in  presence 
of  this  serum.  The  preventive  substance  finds  in  the  leucocytes 
the  bactericidal  substance  there  present.  From  this  moment  the 
leucocyte  has  acquired  a  powerful  and  specific  bactericidal  prop- 
erty. If  it  takes  up  vibrios  it  can  destroy  them ;  but  if,  for  any 
reason,  it  should  suffer  injury,  it  will  liberate  the  bactericidal  sub- 
stance into  the  surrounding  fluid  and  so  bring  about  an  extra- 
cellular destruction  of  the  vibrio.  If  we  consider  the  facts  from 
this  point  of  view,  as  seems  justifiable,  we  must  admit  that  in 
addition  to  cellular  stimulation  there  is  present  a  purely  chemical 
phenomenon  that  may  be  repeated  in  vitro  and  which  is  of  great 
importance  in  passive  immunity.  This  phenomenon  is  the  union  of 
the  two  substances  that  produce  a  bactericidal  power  either  in  the 
animal  or  in  the  test  tube.  An  immunity  of  this  sort  brought  about 
in  this  manner  need  not  be  very  powerful,  and  it  is  not  necessary 
for  its  formation  that  a  profound  cellular  change  should  be  caused 
as  would  be  essential  for  its  persistence.  Passive  immunity  is 
essentially  ephemeral  and  evidence  of  it  is  not  long  shown  by 
the  cell.  As  soon  as  the  preventive  substance  is  eliminated  the 
leucocytes  will  have  no  more  than  a  limited  bactericidal  property, 
and  the  refractory  condition  so  rapidly  obtained  will  be  quite  as 
rapidly  lost. 


IV.  ON  THE  MODE  OF  ACTION  OF  PREVENTIVE  SERA  * 

BY  DR.  JULES   BORDET. 

PREPARATEUR   AT  THE   PASTEUR   INSTITUT. 
(From  the  Laboratory  of  Professor  Metchnikoff.) 

The  properties  of  the  blood  serum  of  immunized  animals  are 
being  attentively  studied.  While  it  is  still  rather  difficult  to  explain 
the  antitoxic  properties  in  certain  sera,  our  knowledge  of  the  pre- 
ventive power  of  serum,  that  is  to  say  the  property  of  immune  serum 
to  protect  animals  against  the  invasion  of  bacteria,  has  recently 
been  greatly  increased.  In  the  present  article  we  shall  deal,  not 
only  with  certain  new  facts,  but  shall  also  review  the  conceptions 
we  have  held  and  refer  to  the  various  opinions  of  other  writers  on 
these  subjects.  We  must  repeat,  then,  not  only  those  facts  which 
we  personally  have  demonstrated,  but  also  the  researches  of  other 
observers,  notably  of  Pfeiffer  and  of  Gruber.  We  shall  frequently 
refer  to  the  work  of  Metchnikoff  |  whose  experience  and  advice  is 
of  such  signal  value  to  those  who  have  the  opportunity  of  working 
with  him. 

The  attention  of  many  observers  has  naturally  been  directed  to 
those  substances  that  are  harmful  for  bacteria  which  the  animal 
body  uses  in  its  struggle  against  infections,  and  the  presence  of 
which  may  be  easily  demonstrated,  particularly  in  animals  immu- 
nized against  the  cholera  vibrio.  In  such  animals  the  serum  is 
endowed,  not  only  with  a  preventive  power  but  also  with  a  distinct 
bactericidal  power  for  Koch's  vibrio.  One  of  the  chief  points  of 
disagreement  is  whether  this  bactericidal  substance  is  uniformly 
dissolved  in  the  body  fluids  during  life  or  is  confined  to  cells.  The 
action  of  preventive  sera  on  bacteria  in  vitro  has  been  carefully 

*  Sur  le  mode  d'action  des  serums  preVe"ntifs.  Annales  de  1'Institut  Pasteur, 
1896,  X,  193. 

t  Particularly  the  article:  Reche"rches  sur  la  destruction  extracellulaire  des 
bacte"ries,  Annales  de  1'Institut  Pasteur,  June,  1895. 

81 


82  STUDIES  IN   IMMUNITY. 

studied.  Furthermore,  attempts  have  been  made  to  gain  an  insight 
into  the  mechanism  of  the  immunity  conferred  by  serum,  and  to 
determine  the  relation  between  this  kind  of  immunity  (passive 
immunity)  and  the  immunity  caused  by  repeated  injections  of 
bacteria  (active  immunity).  In  these  discussions  the  question  has 
repeatedly  recurred  as  to  the  part  which  is  played  in  animal  pro- 
tection by  the  body  fluids  on  the  one  hand  and  by  the  living  cells 
on  the  other. 

I.   THE  LOCALIZATION  OF  THE  BACTERICIDAL  SUBSTANCE 
DURING  LIFE. 

If  cholera  vibrios  are  placed  in  the  serum  of  an  animal  immunized 
against  these  bacteria  they  are  destroyed  at  least  partially.  After 
a  certain  time  cultures  from  this  serum  on  artificial  media  are  either 
negative  or  show  very  few  colonies.  We  have  already  given  the 
reasons  that  have  led  us  to  conclude  that  the  bactericidal  substance 
present  in  serum  comes  from  leucocytes.  We  were  forced  to  the 
conclusion  that  during  life  the  bactericidal  substance  is  present  in 
leucocytes  and  that  when  the  white  blood  cells  are  removed  from 
the  blood  vessels  they  liberate  into  the  surrounding  serum  those 
bactericidal  substances  which  they  normally  retain.*  We  based 
our  conclusions  on  the  comparative  study  of  the  bactericidal 
property  of  the  serum  of  a  vaccinated  animal  with  the  edema 
fluid  from  the  same  animal,  and  by  a  comparison  between  the 
destructive  property  of  the  serum  from  whole  blood  and  the  serum 
from  blood  previously  deprived  within  the  body  of  part  of  its 
leucocytes. 

The  means  of  determining  the  existence  of  bactericidal  power 
and  of  estimating  its  intensity  was  simply  to  inoculate  culture  media 
(gelatin)  with  organisms  that  had  been  allowed  to  remain  in  contact 
with  the  body  fluids  mentioned  for  variable  lengths  of  time.  We 
have  also  considered  another  reaction  that  has  been  given  to  inves- 
tigators through  the  observations  of  Pfeiffer. 

Vibrios  and  other  bacteria  may  show  the  harmful  influences  of  a 
bactericidal  substance,  not  only  by  losing  more  or  less  completely 
their  power  to  develop  on  such  artificial  media  as  gelatin,  but  also 
by  the  morphological  change  they  show  when  affected  by  this 

*  See  page  24. 


ON  THE   MODE  OF  ACTION  OF  PREVENTIVE  SERA.          83 

harmful  substance.  The  existence  of  bactericidal  power  may  thus 
be  recognized,  and  its  nature  studied  in  detail  by  means  of  a 
morphological  change  evident  on  microscopic  examination. 

As  is  well  known,  Pfeiffer  found  that  when  cholera  vibrios  are 
injected  into  the  peritoneal  cavity  of  a  well-immunized  animal  they 
lose  their  motility  and  undergo  rapid  transformation.  The  same 
phenomenon  occurs  in  a  normal  animal  provided  that  the  emulsion 
of  vibrios  is  mixed  with  a  certain  amount  of  active  preventive  serum. 
Pfeiffer  thought  that  this  phenomenon  of  granular  transformation 
could  occur  only  within  the  animal  body,  and  that  the  substance 
which  caused  this  metamorphosis  of  vibrios  came  from  an  active 
secretion  of  the  endothelial  cells;  he  thought  that  the  leucocytes 
had  no  function  in  the  elaboration  or  in  the  storing  of  these  destruc- 
tive substances.  MetchnikorT  was  then  able  to  show  that  Pfeiffer's 
phenomenon  could  be  produced  in  vitro,  if  the  vibrios  are  mixed 
with  a  small  amount  of  preventive  serum  and  a  little  peritoneal 
lymph  containing  leucocytes.  On  the  other  hand,  the  injection 
into  a  normal  animal  of  vibrios  plus  preventive  serum  in  a  region 
where  there  are  no  leucocytes  (for  example,  a  leg  in  which  an  edema 
has  been  formed)  is  found  to  produce  no  transformation  in  spite  of 
the  presence  of  the  preventive  serum.  Some  time  after  injection, 
when  leucocytes  appear,  transformation  does  take  place,  but  only 
within  the  cells.  We  found,  moreover,  that  the  metamorphosis  of 
the  vibrio  could  be  very  well  brought  about  in  vitro  by  the  action 
of  the  fresh  serum  of  an  immunized  guinea-pig,*  even  when  no  cells 
are  present.  We  found,  too,  that  this  transformation  did  not  take 
place  if,  instead  of  serum  (that  is,  a  fluid  formed  outside  the  body 
that  has  been  in  contact  with  leucocytes  and  therefore  liable  to 
contain  products  of  leucocytic  disintegration),  we  used  edema  fluid 
from  the  same  animal;  in  other  words,  a  fluid  separated  within  the 
animal  body  and  almost  entirely  free  from  leucocytes. 

Aqueous  humor  and  various  secretions  from  an  animal  immunized 
against  the  cholera  vibrio  also  fail  to  produce  a  transformation  of 
the  organism. 

It  was  also  shown  that  other  bacteria  (for  example,  B.  typhosus, 
B.  coli  and  B.  pyocyaneus)  "that  have  been  found  to  be  suscep- 

*  And  also  in  a  mixture  of  fresh  normal  serum  with  a  small  amount  of  pre- 
ventive serum. 


84  STUDIES  IN  IMMUNITY. 

tible  to  granular  transformation  in  vaccinated  animals  even  outside 
the  cells  may  show  the  same  change  in  normal  animals.  But  in  the 
latter  case  the  transformation  occurs  only  in  those  places  where  the 
bactericidal  substance  is  most  concentrated,  that  is  to  say,  within 
the  phagocyte."  We  may  add  that  if  we  inject  B.  coli  into  a 
guinea-pig  with  preventive  serum  we  find  that  granular  transforma- 
tion takes  place  within  the  leucocytes  even  when  the  bacteria  in  the 
surrounding  fluid  are  unaffected. 

These  facts  indicate  very  clearly  the  superiority  in  bactericidal 
power  of  the  white  corpuscles  over  the  body  fluids,  as  is  shown  by 
a  production  of  granules.  They  show,  moreover,  that  the  bacteri- 
cidal power  in  an  exudate  owes  its  origin  entirely  to  the  leucocytes 
in  the  fluid.  We  must  consider  why  this  essential  function  of 
leucocytes  in  the  elaboration  of  bactericidal  substance  has  been 
so  much  doubted  and  is  still  denied  by  many  observers,  and  why 
the  activity  of  the  body  fluids  in  destroying  bacteria  has  seemed 
in  certain  cases  superior  to  the  cell  activity.  It  is  due  simply  to 
the  following  fact :  in  well-immunized  animals  a  very  small  dose  of 
bactericidal  substance  is  sufficient  to  cause  a  granular  change  in 
a  large  number  of  vibrios.*  A  very  few  leucocytes  endow  the 
exudate  with  a  bactericidal  power  which  can  produce  the  meta- 
morphosis of  a  surprising  number  of  bacteria. 

On  the  other  hand,  a  large  supply  of  leucocytes  is  necessary  to 
effect  the  phagocytosis  of  a  moderate  number  of  bacteria.  When 
vibrios  are  injected  into  the  peritoneal  cavity  the  few  leucocytes 
present  suffice  to  give  the  peritoneal  fluid  a  very  distinct  bactericidal 
activity,  but  as  they  are  few  in  number  they  cannot  take  up  many 
organisms.  For  this  reason  the  attention  of  the  observer  is  imme- 
diately monopolized  by  the  extracellular  bactericidal  activity,  so 
that  he  overlooks  the  essential  importance  of  the  cells  present  in  the 
fluid.  Later  on  the  leucocytes  come  .up  in  large  numbers  and 
phagocyte  the  intact  or  altered  vibrios  completely  and  then  it  is 
quite  impossible  even  on  the  most  superficial  examination  to  over- 
look the  importance  of  their  protective  function.  In  short,  extra- 
cellular transformation  of  vibrios  in  the  peritoneal  cavity  is  due  to 

*  Two  or  three  platinum  loops  of  the  serum  of  one  of  our  vaccinated  guinea- 
pigs,  when  mixed  with  a  drop  of  emulsion  of  vibrios,  cause  a  generalized  meta- 
morphosis, and  yet  our  serum  is  not,  as  a  rule,  as  active  as  Pfeiffer's. 


ON  THE  MODE  OF  ACTION   OF  PREVENTIVE   SERA.          85 

the  chance  of  there  being  sufficient  leucocytes  in  the  cavity  when 
the  injection  is  made  to  cause  the  surrounding  fluid  to  become 
bactericidal,  but  too  few  of  them  to  cause  phagocytosis  on  a  large 
scale.  This  is  the  essential  condition  for  the  production  of  Pfeiffer's 
phenomenon.  We  should  expect,  then,  that  if  we  increase  the  num- 
ber of  phagocytes  at  the  point  of  inoculation  we  should  change  the 
appearance  of  the  conflict  between  the  animal  body  and  the  infec- 
tion; and  this  is  indeed  what  happens.  Metchnikoff  injected  a 
mixture  of  preventive  serum  and  vibrios  into  the  peritoneal  cavity 
of  a  guinea-pig  that  had  received  an  injection  of  bouillon  the  day 
before;  this  previous  injection  caused  an  increase  of  leucocytes,  so 
that  the  vibrios  subsequently  inoculated  encountered  large  numbers 
of  cells.  Under  these  conditions  the  vibrios  were  immediately 
taken  up  without  any  evidence  of  extracellular  morphological 
change.  A  morphological  change  goes  on,  to  be  sure,  but  within 
the  phagocyctes  where  all  the  bacteria  are  to  be  found.*  We 
have  already  shown  that  vibrios  are  immediately  phagocyted  when 
injected  into  the  circulation  of  vaccinated  animals.  On  the  other 
hand,  if  the  vibrio  is  injected  into  a  region  deprived  of  leucocytes, 
for  example,  subcutaneously,  or  into  an  edema  area,  no  extracellular 
change  is  found.  Phagocytes  come  up  gradually  and,  as  soon  as 
they  appear  in  the  infected  region,  they  take  up  the  bacteria  which 
have  remained  normal.  Phagocytosis  under  these  conditions  occurs 
as  the  initial  phenomenon,  beginning  with  the  arrival  of  the  cells, 
and  dealing  with  intact  bacteria. 

The  experiments  which  we  have  just  reviewed  as  well  as  the 
conclusions  that  we  have  drawn  from  them  have  given  rise  to 
objections  which  we  must  consider.  Pfeiffer  admits  that  vibrios 
are  changed  in  vitro  by  a  mixture  of  fresh  guinea-pig  serum  and 
preventive  serum,  but  he  thinks  that  this  effect  on  the  vibrios  is 
only  passing  and  not  equal  in  bactericidal  force  to  that  which  occurs 
in  the  animal  body.  We  cannot  share  this  opinion.  If  vibrios  are 
injected  into  the  peritoneal  cavity  of  an  immunized  guinea-pig, 
transformation  allowed  to  take  place,  and  some  of  the  exudate  is 
then  withdrawn,  it  is  found  on  placing  it  in  the  incubator  that  the 

*  We  do  not  understand  how  Gruber  could  use  this  experiment  to  contest  the 
origin  of  the  bactericidal  substance  from  the  polynucle'ar  leucocytes.  (Wiener 
klinische  Wochenschrift,  1896,  No.  12,  p.  207.) 


86  STUDIES  IN   IMMUNITY. 

organisms  grow  out  again  quite  as  well  as  when  the  phenomenon 
has  been  produced  in  a  serum  mixture  in  vitro.* 

Pfeiffer's  objection,  then,  is  untenable.  As  a  matter  of  fact,  the 
power  of  transforming  vibrios  is  very  marked  in  immune  serum. 
There  is  nothing  astonishing  in  the  fact  that  after  a  certain  time 
the  bactericidal  properties  are  exhausted  and,  with  in-vitro  experi- 
ments when  the  substance  is  used  up,  there  is  no  means  of  renewing 
it.  In  the  animal  body,  however,  the  exudate  keeps  on  increasing 
after  injection.  Moreover,  it  would  not  be  surprising  to  find  that 
the  exudate  is  more  bactericidal  than  serum,  since  even  at  the 
moment  of  injection  it  often  contains  more  leucocytes  than  does  the 
blood.  And  finally,  there  is  every  reason  to  believe  that  the  extent 
of  the  humoral  bactericidal  activity  which  occurs  in  the  peritoneal 
cavity  of  immunized  animals  is  exaggerated.  Certain  vibrios  resist, 
and  either  retain  or  recover  their  motility;  and,  under  the  usual 
conditions  of  Pfeiffer's  experiment,  it  is  not  reasonable  to  speak  of 
an  extracellular  destruction  that  occurs  3  or  4  hours  after  injection 
for  the  simple  reason  that  by  this  time  all  the  organisms,  whether 
altered  or  not,  are  within  leucocytes.  After  this  time  any  further 
destruction  of  the  infective  agent  must  be  attributed,  not  to  the 
fluid,  but  to  the  cells.  Pfeiffer  has  repeated  Metchnikoff's  experi- 
ment of  injecting  into  a  normal  guinea-pig  a  mixture  of  vibrios  and 
preventive  serum  in  an  area  where  edema  has  been  caused  by  venous 
compression.  Under  these  conditions  Metchnikoff  found  that  the 
vibrios  outside  the  cells  were  not  changed  into  granules.  In  this 
experiment  naturally  a  preventive  serum  that  has  not  been  freshly 
obtained  must  be  used,  since  a  fresh  serum  might  of  its  own  accord 
produce  a  metamorphosis  of  the  vibrio.  Pfeiffer  obtained  a  different 
result  from  the  one  noted  by  Metchnikoff,  that  is  to  say,  he  did  find 
an  extensive  extracellular  change  in  the  organisms.  Metchnikoff's 
results,  however,  were  constant.  We  have  found,  moreover,  that  in 
vitro  the  cholera  vibrio  is  unchanged  by  a  mixture  of  preventive 
serum  and  edema  fluid,  whereas  it  is  transformed  by  a  mixture  of 
preventive  serum  and  fresh  normal  serum.  The  unexpected  result 
of  Pfeiffer's  experiment  may  be  due  to  the  fact  that  this  edema  was 
not  pure  plasma,  but  contained  a  certain  amount  of  blood.  The 
punctures  that  have  to  be  made  to  obtain  the  exudate  may  very 

*  This  was  very  clearly  shown  in  Metchnikoff's  experiments. 


ON  THE  MODE  OF  ACTION  OF   PREVENTIVE  SERA.          87 

well  produce  slight  hemorrhages,  and  we  know  that  even  a  small 
amount  of  blood  serum  will  affect  vibrios  in  presence  of  the  pre- 
ventive substance. 

Pfeiffer,  moreover,  was  unable  to  find  the  almost  instantaneous 
occurrence  of  phagocytosis  in  animals  prepared  by  a  previous 
injection  of  bouillon,  under  which  conditions  it  will  be  recalled  the 
vibrios  immediately  encounter  a  large  number  of  phagocytes.  It 
may  be  that  if  one  injects  a  large  amount  of  bacterial  emulsion 
at  a  low  temperature  a  momentary  paralysis  of  the  phacocytes 
may  be  caused  and  an  extracellular  transformation  may  therefore 
occur.  It  does  not  seem  evident  that  Pfeiffer's  conditions  were 
essentially  different,  which  fact  renders  his  results  still  more  inex- 
plicable, as  we  regularly  obtain  rapid  phagocytosis  under  these 
conditions. 

Pfeiffer  mentions  the  case  of  a  goat  that  had  been  well  immunized 
and  that  died  of  intoxication  following  a  subcutaneous  injection  of 
vibrios ;  in  this  instance  the  destruction  of  the  culture  was  brought 
about,  according  to  Pfeiffer,  entirely  without  the  aid  of  phagocytes; 
no  white  cells  were  found  at  the  point  of  inoculation,  although  the 
cultures  were  negative.  When  dealing  with  well-immunized  animals 
a  few  scattered  leucocytes  may  be  sufficient  to  give  the  body  fluid 
a  distinct  bactericidal  power.  What  is  more,  it  is  quite  probable 
that  in  a  dead  animal  the  diffusion  of  bactericidal  substances  does 
not  follow  the  laws  that  obtain  in  a  living  animal.  We  know,  indeed, 
that  when  leucocytes  are  taken  out  of  the  animal  body  they  liberate 
certain  substances  more  readily  than  they  do  under  normal  condi- 
tions, and  the  leucocytes  in  a  dead  or  dying  animal  can  scarcely  be 
considered  to  be  under  normal  conditions. 

II.    THE   EFFECT    OF    SERA    ON    BACTERIA    IN    VITRO.       THE 
DIAGNOSIS  OF  BACTERIA  BY  SERUM. 

Before  endeavoring  to  determine  the  mechanism  of  passive 
immunity  more  exactly  we  must  consider  the  exact  effect  of 
serum  on  vibrios  in  vitro.  To  be  perfectly  clear,  we  must  consider 
this  subject  from  the  beginning,  which  will  necessitate  the  repeti- 
tion of  certain  details  that  have  already  been  published. 

In  the  first  place  we  may  recall  that  the  serum  of  an  immunized 
animal  causes  the  granular  transformation  of  the  vibrio  used  for 


88  STUDIES  IN   IMMUNITY. 

immunization,  which  phenomenon  serves  as  an  index  of  the  bac- 
tericidal power  which  this  fluid  possesses.  If  an  emulsion  of  vibrios 
is  mixed  with  preventive  serum,  they  lose  their  motility,  collect 
into  compact  clumps  and  finally  undergo  transformation.*  This 
effect  by  serum  is  rather  markedly  specific,  as  we  shall  consider  in 
detail  later  on. 

Serum  that  has  been  kept  for  some  time  or  has  been  heated  to 
60  degrees  remains  preventive  and  may  still  immunize  animals,  as 
Fraenkel  and  Sobernheimf  were  the  first  to  note,  but  loses  its  bac- 
tericidal power.  Under  these  conditions  it  also  loses,  as  might  be 
imagined  and  as  we  have  pointed  out,  the  property  of  producing 
granular  transformation  of  the  vibrio.  But,  as  we  have  particularly 
emphasized,  the  bactericidal  power  may  be  restored  to  such  serum 
and  it  may  again  become  able  to  transform  vibrios.  Although 

*  It  had  not  been  distinctly  recognized  before  our  experiments  that  preventive 
serum,  when  added  in  very  small  amount  to  an  emulsion  of  the  bacteria  against 
which  the  serum  is  active,  causes  their  clumping  in  little  masses  that  float  in  the 
fluid.  Observations  on  the  clumping  action  of  the  serum  of  an  immunized  animal 
had,  however,  been  made.  Charrin  and  Roger  (DeVeloppement  des  microbes 
pathogenes  dans  le  serum  des  animaux  vaccines,  Societe*  de  Biologic,  1889,  p.  667) 
may  be  credited  with  this  observation.  These  authors  noted  that  "a  culture  of 
B.  pyocyaneus  becomes  clear  and  transparent  when  placed  in  the  serum  of  an 
immunized  animal.  The  organisms  are  collected  in  tiny  clumps  which  may  be 
separated  by  shaking  the  tube,  but  which  fall  again  to  the  bottom  when  left  stand- 
ing; bacilli  placed  in  normal  serum  show  no  such  peculiarity.  In  .the  serum  of 
refractory  animals,  however,  the  appearance  is  very  distinctive;  the  organisms 
are  united  in  chains,  etc.  Finally,  these  elements  tend  to  collect  together  and, 
instead  of  floating  freely,  as  do  normal  bacilli,  they  pile  up  in  little  clumps,  which 
explains  the  clotted  appearance  of  the  culture." 

Later  on  Metchnikoff  (Annales  Pasteur,  1891,  p.  473  et  474)  noted  the  same 
phenomenon  with  the  vibrio  Metchnikovi;  he  found  that  this  organism  develops 
on  the  blood  and  serum  of  non-vaccinated  guinea-pigs  as  it  does  in  any  ordinary 
fluid  medium.  In  the  serum  of  immunized  animals,  on  the  contrary,  the  culture 
shows  definite  clots  that  float  about  in  a  clear  fluid  on  agitation  and  then  fall  to 
the  bottom  of  the  tube.  He  noted  the  same  fact  with  the  organism  of  pneumonia, 
and  his  observation  was  confirmed  two  years  later  (Annales  Pasteur,  1893)  by 
Issaef,  who,  later  still,  in  collaboration  with  Ivanhoff,  found  that  the  same  phe- 
nomenon occurred  with  a  vibrio  discovered  by  the  latter. 

Although  Metchnikoff  was  inclined  to  regard  these  phenomena  of  clumping 
as  of  general  significance,  he  made  no  definite  statement  to  this  effect,  because  the 
phenomenon  did  not  occur  in  cultures  of  the  organism  of  pneumo-enteritis  on 
adding  the  serum  of  animals  immunized  against  it  (Annales  de  Tlnstitut  Pasteur, 
1892). 

t  Hygienische  Rundschau,  1894. 


ON  THE  MODE  OF  ACTION  OF  PREVENTIVE  SERA.          89 

heated  immune  serum  contains  only  the  preventive  substance,  its 
strong  antiseptic  power  against  the  vibrio  may  be  restored  by 
mixing  with  it  fresh  normal  serum,  which  in  itself  is  only  faintly 
bactericidal.  This  simple  experiment  led  us  to  the  rather  para- 
doxical conclusion  that' " two  sera,  neither  of  which  is  distinctly 
bactericidal,  form  a  mixture  which  has  marked  antiseptic  proper- 
ties against  vibrios." 

Only  a  very  small  amount  of  preventive  substance  is  necessary  to 
endow  the  preformed  substance  of  normal  serum  with  great  activity 
and  with  specificity.  Normal  serum  heated  to  55  degrees  loses  its 
power  to  form  a  bactericidal  mixture  with  preventive  serum. 
Edema  fluid,  aqueous  humor,  etc.,  from  a  normal  guinea-pig  differ 
from  normal  serum,  since,  mixed  with  preventive  serum  and  an 
emulsion  of  vibrios,  they  cause  no  metamorphosis.  The  preventive 
substance  in  immunized  animals  passes  more  rapidly  into  the  edema 
fluid  than  does  the  bactericidal  substance  as  is  shown  by  the  fact 
that  the  edema  fluid  of  a  vaccinated  guinea-pig,  which  does  not 
cause  granular  transformation,  will  produce  it  on  the  addition  of  a 
small  amount  of  fresh  serum. 

We  were  led  to  the  conclusion  that  the  intense  bactericidal  prop- 
erty of  immune  serum  is  due  to  the  combination  of  two  substances ; 
one,  the  specific  preventive  substance  which  resists  heating  to  60 
degrees  or  even  more;  the  other,  the  bactericidal  substance  properly 
speaking,  which  is  not  specific  and  in  itself  is  only  slightly  active  and 
is  present  in  normal  as  well  as  in  immunized  animals.  This  latter 
substance  is  sharply  differentiated  by  the  fact  that  it  is  destroyed 
by  keeping  for  any  length  of  time,  or  by  heating  to  55  degrees.  The 
serum  of  vaccinated  animals  when  fresh  contains  both  substances ;  it 
may  easily  be  deprived  of  one  of  them  by  heat,  but  the  original  prop- 
erties of  the  serum  may  be  restored  synthetically  by  adding  to  it 
the  part  that  has  been  lost,  that  is  to  say,  the  bactericidal  substance 
(or  alexin)  of  normal  serum. 

Since  experiment  has  shown  us  that  the  simultaneous  presence  of 
both  substances  is  necessary  to  constitute  bactericidal  power  against 
the  vibrio  we  must  now  consider  what  the  respective  action  of  each 
substance  in  the  mixture  is.  If  we  add  to  an  emulsion  of  vibrios 
preventive  serum  alone,  that  is  to  say,  serum  previously  heated  for 
half  an  hour  to  60  degrees,  we  find  that  the  organisms  remain 


90  STUDIES  IN   IMMUNITY 

morphologically  intact,  but  that  many  of  them  become  motionless 
and  clumped.* 

If  a  mixture  is  made  of  vibrios  and  normal  serum  without  the 
specific  preventive  substance,  certain  bacteria  lose  their  motility  and 
form  round  granules;  this  transformation,  however,  is  only  slight. 

It  is  evident  from  these  facts  that  the  preventive  substance  immo- 
bilizes and  clumps,  and  that  it  becomes  still  more  active  when 
accompanied  by  the  bactericidal  substance.  Of  the  two  substances 
the  normal  bactericidal  substance  is  the  only  one  which  alone  can 
cause  even  a  partial  morphological  change  in  the  organism.  It 
appears  probable  then  "that  the  preventive  substance  exerts  a 
certain  unfavorable  effect  on  the  vibrios,  although  it  is  not  a  real 
antiseptic ;  and  that  this  effect  causes  them  to  react  more  distinctly 
to  the  power  of  the  bactericidal  substance." 

The  origin  of  this  important  bactericidal  substance  which  occurs 
in  normal  as  well  as  in  immunized  animals,  and  also  in  sick  animals 
or  animals  that  have  died  of  various  infections,  is  known.  It  is 
the  substance  found  within  the  leucocytes,  and  which  even  in  normal 
animals  is  sufficiently  concentrated  in  the  cells  to  bring  about  a 
granular  modification  of  organisms  ingested  by  the  phagocytic 
protoplasm.  As  a  result  of  the  coagulation  of  the  blood  it  is  partially 
liberated  in  the  serum,  and  gives  evidence  there  of  its  activity  when 
combined  with  the  preventive  substance,  although  this  activity  is 
necessarily  weakened  by  dilution. 

This  represented  the  status  of  our  knowledge  a  year  ago.  In  a 
series  of  recent  articles  Gruber  has  carefully  repeated  the  majority 

*  It  must  be  noted  that  this  phenomenon  of  immobilizing  and  clumping  is  not 
nearly  so  striking  with  heated  serum  as  with  fresh  serum.  In  heated  serum, 
moreover,  the  vibrios  grow  rapidly,  and  after  they  cease  to  feel  the  clumping 
influence  of  the  serum  spread  diffusely  throughout  the  fluid.  Some  time  ago 
Metchnikoff,  in  his  work  on  the  vibrio  Metchnikovi,  noted  that  the  vibrios  grew 
unclumped  in  the  inflammatory  exudate  of  the  immunized  guinea-pig,  whereas 
they  were  agglutinated  when  inoculated  in  the  serum  of  the  same  animal.  This 
offers  yet  further  analogy  between  the  properties  of  the  exudate  and  the  heated 
serum,  which  we  have  already  found  to  be  very  similar.  Both  fluids  contain 
(provided  they  have  been  taken  from  immunized  animals)  the  preventive  sub- 
stance; but  their  bactericidal  activity  is  very  much  less  than  that  of  intact  serum. 
It  may  also  be  added  that  bacteria  cultivated  in  heated  preventive  serum  undergo 
clumping  and  granular  transformation  when  a  certain  amount  of  normal  serum  is 
added  to  the  culture.  This  addition  restores  to  the  serum  its  original  qualities, 
even  after  it  has  served  as  a  culture  medium. 


ON  THE  MODE  OF   ACTION   OF   PREVENTIVE   SERA.          91 

of  the  experiments  which  we  have  mentioned  and  added  others, 
and  as  we  shall  see  presently  he  has  drawn  certain  conclusions  as 
to  the  mechanism  of  passive  immunity  which  are  in  part  similar 
to  our  own.  This  confirmation  is  gratifying,  but  we  shall  not  con- 
sider further  such  experiments  and  conclusions  as  have  already 
been  mentioned.  We  shall  consider  more  particularly  the  new 
facts  and  interpretations  which  he  has  offered.  First  of  all  Gruber 
finds  that  B.  coli  and  B.  typhosus  react  under  similar  conditions  in 
the  same  way  as  do  the  vibrios,  that  is  to  say,  they  lose  their  motility 
and  become  clumped. 

Gruber  thinks  that  a  swelling  and  viscosity  of  bacteria  will 
explain  their  clumping  by  preventive  sera.  He  thinks,  moreover, 
that  this  viscosity  actually  produces  the  collection  and  adhesion 
of  separated  organisms  into  clumps.  On  this  account  Gruber  calls 
those  substances  that  produce  clumping  "agglutinins."  However 
fitting  this  term  may  be,  we  must  consider  first  whether  the  swell- 
ing and  viscosity  of  bacteria  can  explain  a  collection  and  clumping 
of  disseminated  micro-organisms.  It  is  quite  conceivable  that  vis- 
cosity may  prevent  the  separation  of  bacteria  already  clumped, 
but  it  is  not  quite  so  clear  how  this  change  can  bring  them  together, 
particularly  when  we  are  dealing  with  non-motile  organisms. 
Tetanus  bacilli  and  streptococci  are  clumped  by  sera;  and  motile 
organisms  (such  as  the  cholera  vibrio)  are  clumped,  even  when  they 
have  been  killed  with  chloroform  and  so  lost  their  motility. 

When  cholera  vibrios  are  mixed  with  heated  cholera  serum 
clumping  occurs,  but  no  granular  transformation  is  to  be  noted. 
The  clumping,  however,  occurs  very  well,  even  when  serum  that  has 
been  kept  for  some  time  is  used ;  clumping,  moreover,  occurs  very 
rapidly,  whereas  the  metamorphosis  takes  some  time.  Vibrios  that 
have  been  rapidly  brought  together  into  definite  masses  when  colored 
by  the  usual  stains  show  no  distinct  alterations.* 

It  is  evident  then  that  the  viscosity  of  clumped  bacteria  cannot 
be  directly  demonstrated  and  consequently  must  be  regarded  as 
purely  hypothetical.  This  idea  of  a  surface  modification  of  bacteria 
which  Gruber  regards  as  certain  becomes  less  admissible  when  we 

*  When  about  to  send  these  pages  to  the  press  we  found  that  Pfeiffer  has  noted 
that  same  fact  in  the  last  number  of  the  Deutsche  medicinische  Wochenschrift, 
April  9,  1896. 


92  STUDIES  IN   IMMUNITY. 

study  the  clumping  produced  by  specific  sera  in  organisms  other 
than  the  vibrios.  If,  for  example,  we  examine  the  clumps  of  tetanus 
bacilli  which  are  well  formed  by  the  action  of  an  antitoxic  horse 
serum,  we  find  that  the  bacteria,  although  collected  in  small  masses, 
do  not  adhere  well.  A  similar  fact  is  suggested  on  examination  of 
stained  preparations  of  masses  of  vibrios.  The  preparations  made 
from  a  clumped  culture  of  tetanus  suggest  in  appearance  a  handful 
of  pins  that  has  been  carelessly  thrown  on  a  table.  The  clearly 
defined  stiff  rods  cross  each  other  in  all  directions  without  giving 
evidence  of  an  intimate  contact,  and  with  rather  large  spaces 
between  them.  The  appearance  of  the  bacilli  is  absolutely  normal, 
even  when  they  have  remained  for  a  long  time  in  the  serum.  An 
examination  of  clumped  streptococci  gives  similar  results.  Clump- 
ing may  be  noted  also  in  cells  other  than  bacteria.  Red  blood  cor- 
puscles, for  example,  may  show  it  very  distinctly.  If  a  small  amount 
of  rabbit  serum  is  added  to  guinea-pig  serum  containing  a  few  blood 
corpuscles  the  latter  very  rapidly  undergo  a  curious  modification; 
instead  of  remaining  scattered  uniformly  throughout  the  fluid  they 
collect  in  perfectly  definite  clumps  which  stand  out  as  little  red 
points  in  a  clear  liquid. 

But  if  instead  of  using  rabbit  serum,  we  add  the  serum  of  one 
guinea-pig  to  the  corpuscles  of  another,  no  change  takes  place  in  the 
uniform  turbidity  of  the  fluid.  Horse  serum  when  added  to  red 
blood  corpuscles  of  the  guinea-pig  or  of  the  rabbit  clumps  them 
energetically.  In  a  similar  manner  rat  serum  clumps  rabbit  cor- 
puscles and  vice  versa;  goat  serum  produces  the  same  effect  on 
guinea-pig  corpuscles.  These  facts  would  indicate  that  as  a  general 
rule  red  blood  corpuscles  are  clumped  by  serum  from  a  different 
animal  species. 

It  may  be  added  that  the  cholera  vibrio,  when  cultivated  in 
guinea-pig  serum  containing  corpuscles,  clumps  them.  It  would  be 
interesting  to  ascertain  whether  red  blood  corpuscles  subjected  to 
the  effect  of  foreign  serum,  or  taken  from  an  infected  animal,  still 
show  normal  osmotic  properties  and  act  in  the  usual  manner  with 
salt  solutions. 

There  is  a  striking  analogy  between  the  clumping  of  red  blood 
cells  by  a  foreign  serum  and  the  effect  of  preventive  serum  on  bac- 
teria. If  we  imagine  that  bacteria  swell  up  and  become  viscous 


ON  THE  MODE  OF    ACTION  OF    PREVENTIVE   SERA.          93 

because  we  find  them  clumped,  we  should  draw  the  same  conclusion 
as  to  the  clumping  of  corpuscles.  If,  however,  we  examine  the 
clumps  of  red  blood  corpuscles  produced  by  horse  serum,  it  is  found 
that  they  show  no  distinct  difference  from  normal  corpuscles  whether 
examined  fresh  or  in  stained  preparations.  If  two  or  three  drops 
of  such  clumped  cells  are  heated  on  a  slide  to  60  degrees,  the  cor- 
puscles separate  quickly  as  the  temperature  rises;  on  cooling  again 
the  clumping  reappears  and  disappears  again  when  reheated;  and 
this  procedure  may  be  repeated  two  or  three  times.  A  rise  in  tem- 
perature is  found  to  produce  the  same  effect  on  clumped  bacteria, 
although  in  this  case  the  phenomenon  is  not  so  readily  observed.* 
Corpuscles  or  bacteria  that  have  been  separated  from  the  clumps 
by  means  of  heat  are  morphologically  and  tinctorially  normal.  The 
hypothesis  that  cells  such  as  bacteria  or  red  blood  cells  undergo 
some  modification  when  subjected  to  specific  serum  has  little  prob- 
ability. This  hypothesis,  moreover,  we  may  repeat,  would  account 
for  the  persistence  of  the  clumps,  but  not  for  their  formation;  it 
does  not  explain  the  action  of  heat  just  noted  nor  is  it  in  harmony 
with  the  microscopic  appearance  of  the  organisms,  which,  although 
clumped,  show  no  visible  alterations.  These  clumping  phenomena 
seem  due  rather  to  some  phenomenon  of  molecular  physics.  The 
slightest  effects,  as  we  know,  may  cause  chemical  precipitates 
which  have  remained  uniformly  suspended  in  a  fluid  to  fall  to  the 
bottom. 

It  is  probable  that  serum  acts  on  bacteria  by  changing  the  relations 
of  molecular  attraction  between  the  bacteria  and  the  surrounding 
fluid.  As  far  as  the  effect  of  heat  is  concerned  it  may  be  explained 
by  the  well-known  effect  of  temperature  changes  on  molecular 
phenomena,  as,  for  example,  on  the  superficial  tension  of  fluids. 

This  point  of  view  would  imply  that  bacteria  in  suspension  act 
as  inert  particles  when  clumped  by  a  serum.  There  can  be  no 
question  of  active  participation,  as  we  know  that  cholera  vibrios 
killed  by  chloroform  may  still  be  clumped. 

The  second  point  which  has  been  considered  by  Gruber  and 
Durham,  f  and  which  they  have  explained  in  a  rather  peculiar  man- 

*  Heat  has  little  effect  on  clumps  of  streptococci,  but  this  is  due  to  the  fact 
that  the  chains  of  organisms  are  so  interlaced  as  to  hold  together. 

t  Eine  neue  Methode  zur  raschen  Erkennung  der  Choleravibrio  und  des  Typhus- 
bacillus.  Miinchener  med.  Wochenschrift,  March  31,  1896. 


94  STUDIES  IN  IMMUNITY. 

ner,  is  the  question  of  the  specificity  of  sera  and  the  value  of  this 
specificity  as  a  means  of  certain  diagnosis  of  bacterial  species.  The 
diagnostic  method  founded  on  the  instance  of  specificity  offered 
by  Pfeiffer  is  well  known.  The  serum  of  an  animal  vaccinated 
against  the  cholera  vibrio,  for  example,  when  injected  together  with 
a  culture  of  the  true  vibrio  into  the  peritoneal  cavity  of  a  normal 
guinea-pig  causes  loss  of  motility  and  then  rapid  transformation  of 
the  vibrios  into  roundish  granules.  We  are  safe  in  concluding  that 
all  vibrios  which  give  this  reaction  in  presence  of  an  anticholera 
serum  are  true  cholera  vibrios.  According  to  Pfeiffer,  all  the  true 
cholera  vibrios  do  act  in  the  same  way,  which  fact  gives  us  a  means 
of  distinguishing  Koch's  vibrio  from  similar  vibrios  and  from  other 
species  of  bacteria. 

This  method,  however,  has  not  appeared  to  all  to  be  absolutely 
certain.  When  we  say,  indeed,  that  sera  are  specific  and  that  one 
may  utilize  this  specificity  as  a  means  of  diagnosis  two  distinctly 
independent  facts  are  implied,  each  one  of  which  demands  a  separate 
demonstration.  It  means,  in  the  first  place,  that  the  animal  body 
during  vaccination  acquires  certain  new  properties  which  are  in  a 
direct  and  intimate  relation  to  the  kind  of  bacteria  that  have  been 
used  in  immunization.  It  would  seem  that  this  fact  has  already 
been  justified  by  experiment ;  we  have  found,  for  example,  that  the 
serum  of  a  guinea-pig  vaccinated  against  cholera  is  specifically 
bactericidal  for  the  true  cholera  vibrios  and  has  little  or  no  effect 
on  vibrios  that  are  known  to  differ  from  the  cholera  vibrios,  for 
example,  the  vibrio  Metchnikovi.*  But  it  is  at  the  same  time 
asserted  that  bacteria  which  have  once  reacted  in  a  positive  or 
negative  manner  to  a  given  preventive  serum  will  thereafter  always 
act  in  the  same  way  whatever  may  be  their  subsequent  condition 
of  existence.  This  statement  implies  that  there  are  no  transitional 
forms  between  organisms  which  react  positively  on  the  one  hand  and 
those  which  act  negatively  on  the  other;  such  a  lack  of  transitional 
forms,  indeed,  is  necessary  to  render  the  method  precise.  This 
conception,  which  tends  to  establish  distinct,  insuperable  limitations 

*  These  facts  were  demonstrated  first  by  Pfeiffer.  We  had  previously  noted 
a  similar  phenomenon,  namely,  that  the  serum  of  animals  vaccinated  against  the 
cholera  vibrio  when  injected  into  a  normal  guinea-pig  endows  the  serum  of  this 
animal  with  intense  bactericidal  power  against  the  cholera  vibrio  and  against 
this  vibrio  only. 


ON  THE  MODE  OF  ACTION  OF  PREVENTIVE  SERA.          95 

between  bacterial  species  that  are  so  difficult  to  define  and  separate, 
would  scarcely  appear  to  be  in  harmony  with  our  present -knowledge 
of  the  origin  of  species.  Moreover,  we  know  that  the  vibrio  of 
Massaouah,  which  is  certainly  a  cholera  vibrio,  does  not  show  the 
granular  change  with  cholera  serum.  The  method,  however,  in 
many  instances  may  be  of  real  value  and  aid  considerably  in 
confirming  the  diagnosis. 

Instead  of  injecting  vibrios  and  serum  into  the  peritoneal  cavity 
of  an  animal,  there  is  a  simpler  method,  which  we  have  described  as 
a  modification  of  the  Pfeiffer  diagnostic  procedure,  and  which  con- 
sists in  making  a  mixture  of  the  vibrio  and  fresh  preventive  serum 
in  vitro  (in  case  the  preventive  serum  is  not  fresh,  fresh  normal 
serum  may  be  added).  If  the  organism  is  susceptible  to  the  action 
of  the  serum,  in  other  words,  if  it  belongs  to  the  strain  against  which 
the  animal  that  has  furnished  the  serum  has  been  vaccinated,  it 
will  lose  its  motility  by  clumping  and  show  granular  transformation 
as  already  described.  We  described  this  procedure  in  vibrios  only, 
whereas  Pfeiffer  has  applied  his  method  not  only  to  vibrios,  but  to 
the  colon  bacillus  and  the  typhoid  bacillus  as  well. 

Gruber  and  Durham  make  a  diagnosis,  not  only  between  vibrios, 
but  even  between  B.  coli  and  B.  typhosus,  using  as  the  single 
criterion  the  immobilizing  and  clumping  effect  which  each  specific 
serum  shows  for  its  respective  micro-organism.  For  example,  it  is 
assumed  that  a  suspension  contains  typhoid  bacilli  if  the  organisms 
in  it  are  clumped  by  antityphoid  serum. 

As  far  as  vibrios  are  concerned  Gruber  and  Durham's  method  is 
only  a  mutilation  of  our  own  in- vitro  method.  Our  method,  as  we 
have  just  described  it,  gives  three  distinct  indications  of  the  reaction 
of  the  bacteria  in  question  to  the  serum  that  is  used.  These  indica- 
tions are  loss  of  motility,  clumping,  and  granular  transformation. 
Gruber  and  Durham  pay  no  attention  to  this  latter  important  indi- 
cation and  consider  only  the  first  two.  A  priori  it  would  seem 
impossible  to  have  too  many  distinctive  indications  in  dealing  with 
a  task  so  delicate  as  a  diagnosis  between  two  closely  related  species 
of  bacteria,  and  it  would  seem  that  ignoring  one  of  these  indicat- 
ing signs  would  scarcely  constitute  progress.  In  other  words,  we 
should  consider  all  the  indications  of  specificity  that  our  data  may 
afford  us. 


96  STUDIES  IN  IMMUNITY. 

If  those  substances  in  sera  which  clearly  and  actively  cause  the 
immobilization  and  clumping  of  a  given  bacterial  species  are  to  be 
found  in  the  serum  of  a  vaccinated  animal;  and  if  the  clumping  by 
a  specific  serum  occurs  only  with  bacteria  which  are  similar  to  those 
used  to  vaccinate  the  animal  from  which  the  serum  is  derived,  and 
if,  thirdly,  bacteria  of  the  same  species  always  act  in  a  similar  man- 
ner with  a  given  serum;  we  may  then  regard  as  exact  diagnoses 
based  on  clumping  alone,  without  considering  such  other  characters 
as  granular  transformation.  Not  all  these  conditions,  however,  are 
realized. 

First : — Clumping  of  bacteria  may  be  produced  by  sera  other  than 
specific  preventive  sera;  for  example,  normal  horse  serum  clumps 
cholera  vibrios  energetically.  A  similar,  though  slightly  feebler 
effect,  is  produced  on  the  vibrio  Metchnikovi ;  it  also  happens  fre- 
quently with  B.  tetani,  B.  coli  and  B.  typhosus  and  less  distinctly 
with  the  streptococcus  (the  culture  used  was  very  virulent).*  This 
property  persists  in  horse  serum  that  has  been  kept  or  heated  for 
half  an  hour  to  from  60°  to  62°  C. ;  under  this  latter  condition,  how- 
ever, the  property  is  somewhat  diminished. 

If  two  drops  of  horse  serum  are  added  to  1  c.c.  of  an  emulsion  of 
vibrios  (a  24-hour  agar  culture  suspended  in  20  c.c.  of  normal  salt 
solution)  the  bacteria  fall  rapidly  to  the  bottom  of  the  tube  and 
the  supernatant  fluid  becomes  absolutely  clear  within  two  hours. 
The  clumping  property,  then,  is  well  developed  in  horse  serum,  and 
much  better  developed  relatively  than  is  the  immobilizing  property. 
For  it  is  found  on  examining  hanging  drop  preparations  from  the 
emulsion  of  vibrios  and  serum,  that  there  are  vibrios  which,  although 
brought  together  in  definite  masses,  have  not  entirely  lost  their 
motility;  these  motile  vibrios,  moreover,  frequently  cause  a  move- 
ment or  turning  of  the  entire  mass.  These  facts  may  be  noted  even 
when  a  large  amount  of  serum  is  employed.  But  if  such  mixtures 
have  been  made  according  to  Gruber's  method  in  test  tubes,  the 
deposition  of  bacteria  is  complete,  as  clumps  fall  to  the  bottom  in 
spite  of  their  oscillating  movements.  Although  the  specific  preven- 
tive serum  of  the  immunized  guinea-pig  gives  complete  immobili- 
zation more  easily,  horse  serum  produces  the  same  effect  on  the 

*  Normal    guinea-pig    serum    has    practically   no  effect  on  these  different 
bacteria. 


ON  THE   MODE  OF  ACTION  OF  PREVENTIVE  SERA.  97 

majority  of  the  vibrios  and  causes  very  energetic  clumping.  If  we 
note  the  clarification  of  tubes  containing  an  emulsion  of  cholera 
and  a  very  small  dose  of  specific  serum  we  find  that  it  takes  con- 
siderable time  for  completion.  It  is  partial  at  first,  owing  to  a 
rapid  deposition  of  clumps,  but  proceeds  gradually  by  the  falling  of 
isolated  motionless  vibrios  that  are  not  clumped,  and  therefore  sink 
to  the  bottom  slowly. 

We  have  then  two  sera  which,  although  essentially  different,  have 
similar  properties.  We  might  look  on  the  horse  as  having  naturally 
the  preventive  substance  against  the  cholera  vibrio  and  on  this 
supposition  we  might  postulate  that  the  injection  of  horse  serum 
would  give  guinea-pigs  a  certain  immunity  against  this  organism.* 
But  the  clumping  substance  of  normal  horse  serum  differs  very 
distinctly  from  the  similar  clumping  substance  of  the  immunized 
guinea-pig.  The  first  causes  an  agglutination  of  the  red  blood  cor- 
puscles of  the  normal  guinea-pig,  but  the  second  does  not.  We  may 
conclude,  then,  that  the  property  of  clumping  bacteria  does  not 
belong  exclusively  to  the  specific  preventive  substance,  and  we  can- 
not refer  to  " clumping  substance"  and  " preventive  substance"  as 
synonymous.  All  that  we  can  say  is  that  in  animals  whose  serum 
normally  is  neither  clumping  nor  preventive,  vaccination  may  give 
rise  to  both  properties. 

Second: — If  the  objection  which  we  have  just  outlined  were  the 
only  one,  we  might  use  the  clumping  effect  of  preventive  sera  as  a 
means  of  diagnosis  if  we  took  care  to  use  only  an  immune  serum 
from  animals  in  whose  serum  no  such  property  is  present  before  im- 
munization (the  guinea-pig). 

But  the  effect  which  we  are  considering  is  never  entirely  specific, 
even  in  serum  from  such  animals.  Gruber  himself  mentions  certain 
examples  which  invalidate  the  law  of  specificity.  We  must  con- 
sider this  point  rather  in  detail.  We  find  that  serum  from  guinea- 
pigs  vaccinated  against  the  cholera  vibrio  (Eastern  Prussia)  agglu- 
tinates energetically  the  vibrios  from  Eastern  Prussia,  of  Massaouah 
and  less  completely,  but  very  distinctly,  the  colon  bacillus.  And 

*  This  fact  has  been  noted  by  Pfeiffer  and  we  can  confirm  it.  The  preventive 
power,  however,  is  never  very  strong.  We  may  add  that  the  subcutaneous  injec- 
tion of  3  c.c.  of  horse  serum  in  the  guinea-pig  markedly  increases  the  bactericidal 
property  of  the  animal.  The  increase,  however,  is  not  nearly  so  great  as  that 
caused  by  specific  serum. 


98  STUDIES  IN  IMMUNITY. 

again,  the  serum  of  a  rabbit  vaccinated  against  Massaouah  (which, 
however,  was  not  very  powerful)  clumped  the  Massaouah  organism, 
and  had  only  the  slightest  effect  on  the  vibrio  of  Eastern  Prussia  as 
we  have  already  noted  in  conjunction  with  Mesnil.  It  is,  therefore, 
not  certain  that  in  using  different  sera  we  shall  always  obtain  the 
same  result. 

Third: — A  micro-organism  which  ordinarily  is  clumped  by  a 
given  serum  may  undergo  such  changes  as  to  be  no  longer  distinctly 
affected  by  this  serum.  In  1891  Metchnikoff  found  that  the  vibrio 
Metchnikovi  grown  in  the  exudate  of  an  immunized  animal  might 
either  form  clumps  or  else  grow  as  individual  organisms,  according 
to  the  previous  conditions  under  which  the  culture  had  lived. 
Metchnikoff  and  I  have  recently  convinced  ourselves  that  a  very 
virulent  vibrio  from  Eastern  Prussia,  after  living  for  some  time 
within  the  protoplasm  of  phagocytes,  was  affected  only  very  slightly 
by  serum  from  a  horse  vaccinated  against  cholera.  This  vibrio  had 
been  injected  subcutaneously  into  a  horse  and  the  exudate  with- 
drawn after  phagocytosis  was  complete.  Vibrios  from  the  original 
culture  transplanted  on  agar  were  very  strongly  clumped  by  the 
serum  of  this  animal,  whereas  the  organisms  drawn  from  the  exudate 
of  the  animal  remained  scattered  and  caused  a  permanent  cloudiness 
in  the  fluid.  This  modification  of  the  vibrio,  moreover,  lasts  for 
several  generations. 

When  dealing  with  an  organism  that  has  been  recently  isolated 
we  have  very  indefinite  knowledge  as  to  its  previous  conditions  of 
existence.  It  may  well  be  that  in  nature  certain  influences  change 
organisms  in  such  a  way  that  they  become  insusceptible  to  the 
clumping  action  of  serum.  Since  the  property  of  collecting  bac- 
teria into  clumps  does  not  belong  exclusively  to  specific  preventive 
substances;  and  since  the  clumping  effect  of  a  preventive  serum  is 
not  absolutely  specific;  since  bacteria  are  susceptible  to  changes  in 
respect  to  clumping,  and  their  reaction  to  a  given  serum  is  incon- 
stant and  varying,  it  must  be  admitted  that  suppressing  such  a 
diagnostic  sign  as  the  granular  transformation  does  not  add  to  the 
accuracy  of  this  type  of  investigation. 


ON  THE   MODE  OF  ACTION  OF  PREVENTIVE  SERA.  99 

III.  THE  FUNCTION  OF  CELLS  AND  OF  BODY  FLUIDS  IN  IMMUNITY. 
THE  MECHANISM  OF  PASSIVE  IMMUNITY  AND  ITS  RELA- 
TIONS TO  ACTIVE  IMMUNITY. 

In  the  first  section  of  this  article  we  recalled  to  the  reader  experi- 
ments that  demonstrated  the  respective  activities  of  the  cells  and 
the  body  fluids  on  vibrios  that  had  got  into  the  tissues.  It  was 
mentioned  that  cells  may  act  either  by  liberating  bactericidal  proper- 
ties in  the  surrounding  fluid  or  by  taking  up  intact  or  modified  bac- 
teria. It  was  evident  also  that  of  these  two  modes  of  action  the 
second  is  the  more  important  and  the  only  essential  one.  Phago- 
cytosis takes  place  in  all  cases  and  neither  previous  granular 
change  nor  loss  of  motility  are  necessary  for  its  occurrence.  The 
body  fluids  in  transforming  or  changing  vibrios  act  simply  because 
they  have  received  active  principles  from  the  leucocytes.  Although 
it  is  true  that  the  fluids  may  in  certain  cases  destroy  large  numbers 
of  vibrios,  phagocytosis  nevertheless  occurs  markedly  and  is  the 
ultimate  means  of  destroying  the  infection. 

Neither  the  clumping  nor  immobilizing  of  bacteria  is  to  be  con- 
sidered as  indicative  of  their  destruction  or  as  an  indication  of 
their  elimination  without  phagocytosis,  even  to  the  extent  that 
granular  transformation  is.  We  have  already  shown  that  clump- 
ing of  bacteria  is  no  indication  of  their  alteration,  that  their  sur- 
face has  become  viscous,  nor  that  they  are  swollen  or  incapable  of 
reproduction.  Moreover,  this  phenomenon  is  never  complete  in 
the  animal  body  and,  when  the  transformation  of  vibrios  into 
rounded  bodies  follows  in  the  peritoneal  cavity,  many  granules 
are  seen  that  remain  separate.  Moreover,  the  clumping  power  of 
a  given  serum  bears  no  relation  to  the  resistance  of  the  animal  that 
furnished  the  serum  to  the  bacterium  in  question.  Horse  serum 
clumps  tetanus  bacilli  energetically,  but  the  horse  is  very  suscep- 
tible to  tetanus;  much  more  so,  indeed,  than  is  the  rabbit,  and  yet 
the  serum  of  this  latter  animal  has  no  effect  on  the  tetanus  bacillus. 
The  rat  is  refractory  to  cholera,  but  its  serum  has  no  effect  on  the 
cholera  vibrio. 

An  organism  may  show  most  evident  clumping  and  yet  retain 
its  virulence  completely.  The  pneumococcus  when  grown  on  the 
serum  of  immunized  animals  forms  clots;  such  cultures,  how- 


100  STUDIES  IN   IMMUNITY. 

ever,  as  Issaeff  (Annales  Pasteur,  1893)  has  shown  are  still  very 
virulent. 

As  a  general  thing  cultures  of  pathogenic  organisms  grown  in  the 
serum  of  immunized  animals  remain  virulent  whether  the  organisms 
are  clumped  or  not  (the  pneumococcus  on  the  one  hand  and  the  bacil- 
lus of  hog-cholera  described  by  Metchnikoff  in  1892  on  the  other). 

Horse  serum  and  even  horse  edema  fluid  has  a  remarkable  clump- 
ing effect  on  the  cholera  vibrio.  The  struggle  against  the  vibrios, 
however,  in  the  body  of  this  animal  takes  place  just  as  it  does  in  other 
animals,  namely,  by  means  of  phagocytosis.  If  we  inject  an  agar  cul- 
ture of  a  virulent  cholera  vibrio  subcutaneously  in  a  horse,*  an  influx 
of  leucocytes  occurs.  A  purulent  fluid  may  be  removed  the  next 
day  from  the  area  of  inoculation  and  is  found  to  contain  leucocytes, 
in  the  protoplasm  of  which  are  found  the  organisms  in  various 
stages  of  degeneration;  there  are  no  free  bacteria  present.  The 
inoculation  of  this  pus  on  agar  even  30  hours  after  injection  gives 
an  abundant  culture.  In  other  words,  the  bacteria  have  been 
taken  up  alive  and  eventually  die  within  the  phagocytes,  as  is 
shown  by  the  fact  that  the  animal  rapidly  recovers. 

Does  this  mean  that  the  extracellular  changes  in  bacteria,  the 
most  important  of  which  is  granular  transformation,  is  without 
importance  in  immunity?  In  no  manner;  by  this  means  the  num- 
ber of  invaders  may  be  decreased,  their  development  hindered  and 
time  given  the  animal  to  combat  them  advantageously  by  means 
of  the  leucocytes,  that  have  eventually  come  up,  instead  of  being 
faced  by  an  invincible  number  of  adversaries  and  their  elaborated 
toxic  substances. 

In  order  to  explain  the  mechanism  of  passive  immunity  we  must 
account  for  one  very  important  property  acquired  by  animals 
treated  with  preventive  serum.  To  what  is  the  bactericidal  power 
that  is  given  these  animals  due?  We  have  nothing  essentially  new 
to  add  to  observations  already  made  on  this  subject.  The  mixture 
in  vitro  of  fresh  normal  serum  and  a  small  amount  of  preventive 
serum,  neither  of  which  sera  is  in  itself  bactericidal,  produces  a 
fluid  that  is  strongly  bactericidal.  We  have  repeatedly  emphasized 
this  experiment  because  it  appears  to  us  to  be  the  explanation  of 
the  appearance  of  a  bactericidal  property  in  the  blood  of  passively 

*  The  culture  was  kindly  furnished  us  by  Dr.  Salimbeni. 


ON  THE  MODE  OF  ACTION  OF   PREVENTIVE  SERA.         101 

immunized  animals.  The  study  of  an  infection  in  such  passively 
immunized  animals  shows  that  their  resistance  is  due  to  a  mechan- 
ism similar  to  that  which  we  described  in  June,  1895  as  occurring  in 
vitro.  Bactericidal  power  arises  in  the  animal  body,  just  as  it  does 
in  a  test  tube,  by  the  union  of  two  substances :  a  specific  preventive 
substance,  and  the  normal  bactericidal  substance  present  in  the 
blood,  not  only  of  immunized,  but  also  of  normal  animals.  Sepa- 
rately, each  one  of  these  substances  has  only  the  slightest  effect; 
together,  they  change  the  micro-organism  most  evidently. 

The  normal  animal  has  the  normal  bactericidal  substance.  By 
treating  this  animal  with  immune  serum  we  give  to  it  the  preventive 
substance  which  is  lacking.  How  do  these  two  substances  unite? 
If  the  bactericidal  substance  under  normal  conditions  is  uniformly 
dissolved  in  the  body  fluids.,  the  union  would  take  place  in  these 
fluids.  But  we  know  that  this  bactericidal  substance  is  concen- 
trated in  the  leucocytes.  The  antiseptic  mixture,  then,  must  be 
formed  primarily  by  a  union  within  the  leucocytes  and  can  take 
place  outside  the  cells  only  when  a  certain  amount  of  the  alexin  has 
been  liberated  by  the  cells  into  the  surrounding  fluid. 

Passive  immunity  is  due  then,  at  least  partially,  to  the  chemical 
effect  of  two  preexisting  substances  on  the  vibrios.  One  of  these 
substances  is  present  in  the  animal  before  injection  and  the  other 
comes  from  the  serum  injected.  The  phenomenon  is  chemical 
in  the  sense  that  it  can  occur  without  the  aid  of  any  vital  reaction 
or  any  new  cellular  secretion,  as  we  know  from  the  fact  that  it 
occurs  in  fluids  that  contain  no  cells.  With  the  aid  of  this  new 
means  of  defense,  which  has  been  acquired  without  reaction,  the 
leucocytes  are  more  than  ever  able  to  utilize  their  protecting  power.* 
Passive  immunity  then  may  be  explained,  at  least  in  part,  as  a  pas- 
sive increase  of  the  phagocytic  bactericidal  power.  Gruber  tacitly 
agrees  to  this  explanation,  at  least  so  far  as  the  production  of  a 
bactericidal  property  by  means  of  the  union  of  two  substances  is 
concerned.  It  is,  moreover,  evident  that  he  does  accept  this  explana- 
tion, since  he  has  drawn  similar  conclusions  in  his  recent  articles. 

*  We  do  not  wish  to  imply  that  the  leucocyte  does  not  react  at  all  to  this 
injected  serum,  although  a  reaction  by  the  leucocyte  is  not  indispensable  for  the 
genesis  of  bactericidal  power.  There  is  no  reason  to  suppose  that  the  leucocyte 
may  not  react  by  an  acceleration  of  its  phagocytic  activities. 


102  STUDIES  IN   IMMUNITY. 

To  be  sure  Gruber  does  not  believe  that  union  takes  place  within 
the  protoplasm  of  the  phagocyte,  simply  because  he  does  not  believe 
that  the  phagocyte  is  the  source  of  the  normal  bactericidal  sub- 
stance. He  does  not  bring  any  new  facts  to  bear  on  this  discussion, 
but  simply  accepts  Pfeiffer's  opinion  which  we  have  already  con- 
sidered. 

The  task  of  explaining  the  intimate  changes  which  take  place 
in  bacteria  as  a  result  of  the  activity  of  the  serum  (immobilization, 
clumping,  granular  transformation)  is  far  from  finished.  Gruber, 
to  be  sure,  has  offered  an  explanation  of  clumping,  but,  as  we  have 
already  seen,  it  is  far  from  satisfactory. 

Pfeiffer  has  a  different  point  of  view  on  the  action  of  sera.  He 
thinks  that  the  immunizing  substances  in  immune  serum  are  present 
in  these  sera  in  an  inactive  form,  and  become  active  by  undergoing 
certain  changes  within  the  animal  body. 

In  the  first  place  it  is  certain  that  sera  contain  active  substances, 
since  they  can  produce  the  same  modifications  on  bacteria  in  vitro 
as  in  vivo.  These  modifications,  moreover,  are  exactly  alike,  and 
equal  whether  in  vivo  or  in  vitro.  And,  further,  it  is  easy  to  show 
that  the  preventive  substance  injected  is  simply  diluted  in  the 
blood  of  the  animal  and  does  not  become  more  active. 

The  serum  of  a  guinea-pig  vaccinated  against  the  cholera  vibrio 
acquires  a  property  that  it  did  not  previously  have,  namely,  the 
property  of  clumping  the  cholera  vibrio.  We  know,  moreover,  that 
the  better  a  serum  clumps  the  more  actively  preventive  it  is.  The 
preventive  value,  then,  may  be  measured  by  the  clumping  property. 
If  we  place  in  separate  tubes  a  given  amount  of  an  emulsion  of 
vibrios  and  add  to  each  tube  a  varying  number  of  drops  of  pre- 
ventive serum  we  can  determine  exactly  what  this  clumping  power 
is.  Controls,  of  course,  are  made  with  emulsion  without  preventive 
serum.  After  a  certain  time  the  deposition  of  bacteria  and  the 
clarification  of  the  supernatant  fluid  reaches  its  maximum  (about 
24  hours  when  the  doses  of  serum  are  small).  In  this  way  the 
smallest  amount  of  serum  which  will  cause  either  a  beginning  of 
clarification  or  complete  limpidity  may  be  determined. 

A  guinea-pig  weighing  300  grams  was  bled  and  then  injected  with 
1  c.c.  of  active  preventive  serum;  the  next  day  the  animal  was 
again  bled.  The  serum  obtained  before  injection  had  only  a 


ON  THE  MODE  OF   ACTION   OF   PREVENTIVE  SERA.          103 

faint  clumping  power  for  the  cholera  vibrio,  even  in  a  large  dose. 
The  serum  taken  the  day  after  injection,  however,  clumped  them  dis- 
tinctly, and  it  was  possible  to  compare  its  power  with  that  of  the 
preventive  serum  used  for  injection.  By  means  of  a  series  of  tubes 
it  was  found  that  the  serum  obtained  after  injection  of  cholera 
serum  is  only  one  thirtieth  as  active  as  the  original  cholera  serum. 
For  example,  in  a  tube  containing  1  c.c.  of  bacterial  emulsion  and 
eight  drops  of  this  serum  the  opacity  is  the  same  as  that  in  a  tube 
containing  4  c.c.  of  bacterial  emulsion  and  one  drop  of  anticholera 
serum.*  In  this  experiment  the  same  results  were  obtained  as  if 
the  1  c.c.  of  cholera  serum  had  been  diluted  in  30  c.c.  of  liquid. 
This  volume  corresponds  fairly  well  to  the  amount  of  blood  in  a 
guinea-pig  of  this  weight,  360  grams. 

This  experiment,  moreover,  shows  why  passive  immunity  cannot 
be  transmitted  indefinitely  and  indicates  that  the  active  principles 
of  the  serum  are  simply  diluted  in  the  body  without  acquiring  new 
and  more  energetic  properties.  A  word  might  be  added  about 
active  immunity.  The  preventive  substances,  which  are  so  valuable 
for  animals  injected  with  them,  are  also  evidently  equally  valuable 
for  those  that  have  elaborated  them.  It  must  be  admitted  that 
active  immunity  is  evidenced  by  a  perfection  of  the  bactericidal 
power  of  phagocytes. f  But  active  immunity  has  also  other 
characteristics,  such  as  an  augmentation  of  the  numberof  phagocytes 
and  an  increase  of  chemiotactic  sensitivity  on  the  part  of  leucocytes, 
as  has  frequently  been  recognized  by  Metchnikoff  and  clearly 
evidenced  by  Massart  in  his  experiments.  We  cannot,  therefore, 
consider  the  two  kinds  of  immunity  as  equally  dependent  on  the 
presence  of  a  preventive  substance. 

*  To  be  sure  the  clarification  took  place  more  rapidly,  although  no  more  com- 
pletely, in  the  tube  containing  serum  from  the  passively  immunized  animal. 
This  is  due  to  the  fact  that  the  serum  in  this  tube  was  in  large  amount  and  there- 
fore acted  not  only  as  a  diluted  preventive  substance,  but  also  as  a  normal  serum, 
and  as  is  well  known,  normal  serum  increases  the  clumping  action  of  preventive 
serum.  The  eventual  amount  of  clarification  in  the  two  tubes,  however,  was 
equal. 

t  See  article,  page  8. 


V.  A  CONTRIBUTION  TO  THE  STUDY  OF 
ANTISTREPTOCOCCUS  SERUM  * 

BY  DR.  JULES  BORDET,  PREPARATEUR  AT  THE  PASTEUR 

INSTITUT. 

I. 
STREPTOCOCCUS  INFECTION. 

A.   STREPTOCOCCUS  INFECTION  IN  THE  GUINEA-PIG. 

Before  beginning  the  study  of  antistreptococcus  serum  and  its 
properties  a  consideration  of  the  principal  characteristics  of  strep- 
tococcus infection  and  the  methods  of  reproduction  of  the  strep- 
tococcus in  the  animal  body  is  indispensable.  We  must  therefore 
consider  the  characters  of  the  micro-organism  that  we  have  used  in 
our  experiments  and  describe  briefly  the  general  aspects  of  the 
infection  which  it  causes. 

The  streptococcus  we  have  used  in  the  majority  of  instances  is 
one  whose  virulence  was  increased  by  Dr.  Marmorek  by  methods 
described  in  the  Pasteur  Annals  of  July,  1895.  Marmorek  has 
been  so  kind  as  to  place  his  organism  and  his  serum  at  our  disposal 
for  these  researches  and  we  wish  herewith  to  express  our  thanks  for 
his  kindness. 

The  readers  of  the  Pasteur  Annals  are  already  aware  that  the 
streptococcus  in  question  is  extremely  virulent;  it  kills  rabbits 
in  a  dose  of  a  fraction  of  a  millionth  of  a  cubic  centimeter.  Its 
minimal  lethal  dose  may  be  as  low  as  a  thousand  millionth  of  a 
cubic  centimeter. 

We  have  regularly  used,  as  a  culture  medium  suitable  to  maintain 
the  virulence  of  this  organism,  a  mixture  of  peptonized  bouillon 
and  human  ascitic  fluid,  as  already  described  by  Marmorek.  On 
this  medium  the  organism  grows  rapidly  and  keeps  its  pathogenic 
qualities  through  many  successive  transplantations. 

*  Contribution  &  l'4tude  du  s£rum  anti-streptococcique.  Annales  de  1'Institut 
Pasteur,  1897,  XI,  177. 

104 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  105 

The  infection  caused  by  this  streptococcus,  although  not  varying 
in  general  appearance,  differs  somewhat  in  details  according  to  the 
animal  species  employed. 

Much  larger  doses  are  necessary  to  kill  a  guinea-pig  than  are 
fatal  for  a  rabbit.  The  minimal  intraperitoneal  dose  for  a  medium- 
sized  guinea-pig  sufficient  to  cause  a  generalized  infection  and 
death  of  the  animal  is  generally  0.2  of  a  cubic  centimeter.  The  results 
following  such  an  intraperitoneal  dose  in  a  normal  guinea-pig  are 
as  follows:  By  puncturing  the  abdominal  wall  of  the  animal  and 
withdrawing  a  little  exudate  at  intervals  with  a  small  pointed  tube 
it  is  easy  to  follow  the  'course  of  the  infection. 

A  fatal  dose.  —  Following  the  inoculation  of  the  culture  the  perito- 
neal fluid  is  more  or  less  limpid  and  contains  only  a  small  number  of 
cells.  This  is  the  period  of  phagolysis  which,  as  Metchnikoff  has 
shown,  regularly  follows  the  introduction  of  cultures  into  the 
peritoneal  cavity.*  The  leucocytes  in  this  exudate,  consisting 
generally  of  the  mononuclear  type,  with  admixtures  of  a  few  true 
eosinophiles  and  infrequent  amphophiles,  do  not  long  remain 
scattered  either  because  they  are  destroyed,  as  Metchnikoff  is  in- 
clined to  believe,  or  because  they  adhere  to  the  peritoneal  walls,  as 
Durham  thinks. 

This  period  of  phagolysis  is  usually  short,  particularly  when  the 
amount  of  fluid  introduced  is  inconsiderable,  and  is  followed  by  the 
appearance  of  mononuclear  and  polynuclear  phagocytes.  These 
latter  cells  appear  particularly  during  the  first  hours  of  the  phe- 
nomenon and  increase  rapidly  in  number,  so  as  to  constitute  the 
majority  of  cells  present. 

The  first  polynuclears  arriving  on  the  scene  (say  an  hour  after 
injection)  take  up  a  few  streptococci;  and  a  considerable  number 
of  the  organisms  introduced  are  soon  found  to  be  phagocyted. 
The  mononuclears  also  frequently  take  up  a  good  many  of  them. 
There  are,  however,  among  the  micro-organisms  injected,  certain 
ones  which  are  not  engulfed.  These  latter,  to  be  sure,  are  very  few  if 
the  minimal  lethal  dose  is  employed  and  they  remain  scattered  in 
the  fluid  in  the  midst  of  an  increasing  number  of  cells. 

*  This  phenomenon  of  phagolysis  is  very  slight  if  as  small  an  amount  as  0.2  of 
a  cubic  centimeter  of  culture  is  injected;  but  it  becomes  more  marked  if  the 
amount  of  fluid  injected  reaches  0.5  to  1  c.c. 


106  STUDIES  IN   IMMUNITY. 

The  number  of  phagocytes  gradually  becomes  increased;  the 
cells  become  infinitely  more  numerous  than  is  necessary  to  take  up 
all  the  streptococci  present.  There  always  remain,  however,  cer- 
tain free  bacteria  and  these  bacteria  soon  multiply.  These  organ- 
isms are  differentiated  by  the  fact  that  they  are  surrounded  by  an 
areola  never  present  under  normal  conditions,  which  takes  a  pale 
pinkish-violet  color  with  Kiihne's  blue.  These  organisms  after  2 
or  3  hours  give  rise  to  new  individuals  also  surrounded  by  an 
areola,  similarly  able  to  avoid  engulfing,  and  usually  occurring  in 
the  form  of  diplococci  or  in  short  chains.  The  number  of  these 
new  bacteria,  as  a  rule,  becomes  considerable  in  6  or  7  hours. 
In  an  exudate  taken  at  this  time  we  find  both  a  large  number  of 
cells  and  a  large  number  of  bacteria.  The  majority  of  the  phago- 
cytes, however,  are  empty  and  no  longer  capable  of  capturing  bac- 
teria. We  have  already  shown  in  a  previous  article*  that  such 
leucocytes  not  only  are  not  paralyzed,  but  are,  on  the  contrary,  even 
more  motile  than  usual  and  are  still  able  to  engulf  other  phagocyt- 
able  micro-organisms,  such  as  the  B.  diphtheria?  or  the  B.  proteus 
vulgaris.  If  a  culture  of  Proteus  or  Diphtheria  is  added  to  such  an 
exudate,  the  leucocytes  immediately  take  up  the  new  bacteria, 
although  they  still  refuse  streptococci;  in  other  words,  they  choose 
between  the  species  of  bacteria.  Streptococci,  then,  secrete  a  sub- 
stance which,  although  it  does  not  inhibit  the  influx  of  leucocytes 
into  the  peritoneal  cavity,  affects  adjacent  phagocytes  unfavorably 
and  prevents  them  from  accomplishing  their  protective  engulfing 
function.  We  may  say  that  they  exercise  a  negative  cJiemiotactic  in- 
fluence on  the  phagocytes.  The  number  of  these  cells  increases  until 
it  becomes  enormous  and,  as  a  rule,  the  guinea-pig  dies  in  from  15 
to  20  hours  with  a  purulent  peritonitis  accompanied  by  an  invasion 
of  the  heart's  blood  by  the  bacteria.  The  leucocytes  retain  their 
faculty  of  engulfing  other  micro-organisms  for  a  long  time.  So  far 
as  the  extracellular  streptococci  are  concerned  they  stain  well, 
are  morphologically  normal,  are  surrounded  by  an  areola  and  show 
at  no  stage  the  slightest  evidence  of  degeneration. 

A  non-fatal  dose. — If  a  smaller  amount  of  culture,  for  example, 
0.1  of  a  cubic  centimeter,  is  injected  intraperitoneally  in  a  guinea- 
pig  instead  of  the  minimal  lethal  dose  rapid  and  complete  phagocytosis 

*  See  page  14. 


STUDY   OF  ANTISTREPTOCOCCUS  SERUM.  107 

occurs.  The  streptococci  being  in  too  small  numbers  are  engulfed 
before  they  have  time  to  adapt  themselves  to  the  medium,  and 
acquire  their  intense  repelling  property  for  leucocytes.  Certain 
ones  among  them,  to  be  sure,  resist  and  remain  free  relatively  longer 
than  the  others.  But  as  their  number  is  inconsiderable  in  propor- 
tion to  the  cells  present  they  finally  encounter  leucocytes  which  are 
so  vigorous  that  they  yield  to  them.  The  totality  of  the  strepto- 
cocci injected  are  eventually  contained  within  the  cells.  The 
guinea-pig  recovers  without  any  further  trouble. 

We  have  already  mentioned  that  the  streptococci  that  are  able 
to  protect  themselves  against  the  attack  of  the  guinea-pig  leucocytes 
are  surrounded  by  an  areola  which  takes  a  peculiar  stain.  We 
may  further  note  that  in  those  experiments  in  which  a  sublethal 
dose  of  streptococci  is  employed  the  more  resistant  micro-organ- 
isms, that  is,  those  last  to  be  engulfed,  are  also  usually  those  with  the 
most  marked  areola. 

The  condition  necessary  for  a  fatal  infection. — In  order  that 
streptococci  injected  into  the  peritoneum  of  the  guinea-pig  may 
produce  a  fatal  infection  the  following  condition  must  be  satis- 
fied; when  phagocytosis  by  the  rare  leucocytes  begins,  the  strepto- 
cocci should  be  in  sufficient  quantity  to  permit  the  more  virulent 
of  them  to  remain  outside  the  cells  long  enough  to  become  accus- 
tomed to  the  chemical  composition  of  the  exudate  so  that  they 
and  their  descendants  may  acquire  to  the  highest  degree  the  faculty 
of  remaining  free  amid  the  increasing  leucocytes.  It  would  follow, 
then,  that  if  we  inject  a  fatal  dose  of  streptococci,  not  into  a  normal 
intraperitoneal  cavity  where  the  cells  at  first  are  few  and  little 
adapted  to  phagocytosis,  but  into  a  peritoneal  cavity  rich  in  vigor- 
ous phagocytic  cells  capable  of  bringing  about  a  rapid  engulfing, 
the  bacteria  will  not  have  the  necessary  time  for  adaptation  and 
for  increasing  their  resistance.  This,  indeed,  is  what  happens.  // 
the  number  of  active  phagocytes  in  the  peritoneal  cavity  of  a  guinea- 
pig  is  increased  by  a  previous  injection  of  bouillon,  a  dose  of  strepto- 
cocci equal  to  at  least  twice  the  ordinary  minimal  lethal  dose  may  be 
injected  without  fatal  effect*  We  shall  not  consider  such  an  experi- 
ment for  the  moment,  but  shall  take  it  up  again  when  we  come  to 

*  The  amount  that  can  safely  be  injected  naturally  has  its  limits.  If  as  much 
as  1.5  or  2  c.c.  is  given,  phagocytosis  is  incomplete,  even  in  a  prepared  guinea-pig. 


108  STUDIES  IN   IMMUNITY. 

consider  the  relative  sensitivity  of  the  rabbit  and  the  guinea-pig 
to  the  streptococcus. 

If  something  near  the  minimal  lethal  dose  of  a  virulent  culture 
has  been  injected  into  the  peritoneal  cavity  of  a  guinea-pig  a  prog- 
nosis may  generally  be  given  by  the  presence  or  absence  of  strepto- 
cocci with  an  areola  outside  the  cells.  If,  for  example,  four  hours 
after  infection  the  exudate  contains  a  small  number  of  extracellular 
streptococci  with  areola  and  a  great  many  leucocytes,  the  fate  of  the 
animal  is  doubtful.  If  the  number  of  such  bacteria  is  very  small, 
so  that  they  may  be  found  on  a  slide  only  with  difficulty,  they  fre- 
quently become  the  prey  of  exceptionally  active  phagocytes.  But 
if  their  number  is  at  all  considerable  we  may  feel  sure  that  they  will 
soon  multiply  unrestrictedly. 

The  issue  of  streptococcus  peritonitis  is  thus  soon  indicated  and 
the  outcome  of  the  conflict  between  cells  and  bacteria  is  clear  very  early. 

Mechanism  of  cure.  —  We  may  review  the  evolution  of  strepto- 
coccus peritonitis  in  the  guinea-pig  by  saying  that  the  cure  of  an 
animal  is  due  to  phagocytosis.  The  existence  in  the  bacteria  of  a 
negative  chemiotactic  influence  preventing  the  accomplishment  of 
phagocytosis,  excludes  a  cure.  Since  the  streptococci  that  in  the 
beginning  have  been  able  to  protect  themselves  from  phagocytes 
multiply  outside  the  cells  without  changes  of  morphology  or  color 
reaction  or  any  diminution  in  their  virulence,  there  is  nothing  that 
leads  us  to  suppose  that  when  animals  withstand  an  infection  there  are 
any  factors  in  their  cure  other  than  phagocytosis.  It  may  be  simply 
mentioned  here  that  virulent  streptococci  taken  up  by  the  phago- 
cytes of  the  guinea-pig  are  absorbed  without  losing  their  activity. 

The  injection  of  a  small  amount  of  guinea-pig  peritoneal  exudate 
containing  no  free  streptococci,  but  with  leucocytes  containing 
engulfed  streptococci,  into  a  rabbit  within  four  hours  after  removal, 
kills  as  a  rule  in  24  hours. 

We  have  already  said  that  a  guinea-pig  that  has  received  a  dose 
of  streptococci  small  enough  to  allow  the  bacteria  to  be  entirely 
engulfed  by  phagocytes  recovers  without  any  further  trouble.  As 
a  general  rule,  indeed,  when  complete  phagocytosis  takes  place  the 
animal  is  at  once  out  of  danger  and  soon  recovers.  Within  two  or 
three  days  the  exudate  ceases  to  contain  living  bacteria.  In  a  few 
rare  instances  an  animal  that  has  lived  several  days  without  showing 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  109 

any  free  bacteria  in  its  peritoneal  exudate  and  that  has  apparently 
returned  to  normal  condition  becomes  sick  again  and  undergoes 
a  new  streptococcus  infection.  Under  these  conditions  the  strepto- 
cocci found  in  the  peritoneal  exudate  are  of  a  peculiar  form. 

They  often  occur  in  short  chains  with  beadings  of  unequal  diame- 
ter; chains  are  also  found  here  and  there  of  extraordinary  length  and 
surrounded  by  a  distinct  and  well-marked  areola  differing  consider- 
ably from  the  one  that  ordinarily  encloses  the  streptococcus.  The 
chromogenic  substance  in  certain  of  these  chains  is  a  line  of  nearly 
regular  granulations  as  is  usual  in  the  streptococci,  but  in  other  larger 
chains,  the  chromogenic  substance  appears  to  be  more  indefinite 
and  at  intervals  shows  indefinitely  walled  off  and  poorly  stained 
sac-like  dilatations.  We  repeat  that  these  cases  of  fatal  relapse  in 
apparently  cured  guinea-pigs  occur  only  rarely  and  at  certain  definite 
stages  of  the  infection.  It  is  probable  that  the  culture  that  brings 
about  these  delayed  reinfections  is  possessed  of  peculiar  qualities 
of  resistance  not  ordinarily  present. 

Larger  doses  (0.5  to  2  c.c.)  are  necessary  when  injected  subcutan- 
eously  to  cause  a  fatal  infection  in  a  guinea-pig. 

B.    STREPTOCOCCUS  INFECTION  IN  THE  RABBIT. 

As  we  have  just  seen,  a  dose  of  about  0.2  of  a  c.c.  is  the  minimal 
amount  of  bacteria  necessary  to  produce  a  fatal  peritonitis  in  the 
guinea-pig.  This  is  an  infinitely  larger  amount  than  is  necessary 
to  cause  death  in  the  peritoneal  cavity  of  a  normal  rabbit.  The 
streptococcus  finds  a  very  favorable  culture  medium  in  the  clear 
peritoneal  fluid  of  this  animal  which  contains  only  few  leucocytes. 
The  streptococci  injected  into  the  rabbit  are  usually  scattered  and 
within  half  an  hour  become  surrounded  by  an  areola  not  ordinarily 
present.  It  is  evident  that  the  production  of  this  areola  is  due  to 
the  activity  of  the  bacterial  secretions,  as  its  color  changes  as  the 
infection  proceeds. 

Shortly  after  inoculation  the  areola  is  seen  as  a  distinctly  out- 
lined zone  which  does  not  color  with  Kuhne's  blue  and  consequently 
appears  whitish  against  a  bluish  background.  In  one  and  a  half 
to  two  hours  it  begins  to  take  a  pale  violet  or  pink  color  that  sub- 
sequently becomes  darker. 

The  original  injected  streptococci  may  take  on  this  areola,  but 


110  STUDIES  IN   IMMUNITY. 

it  is  usually  more  distinct  and  more  deeply  colored  in  those  cocci 
generated  in  the  exudate. 

Leucocytes  soon  appear  in  the  peritoneum  following  injection 
of  the  streptococci.  After  an  hour  or  two  there  are  present  some 
mononuclears  and  many  polynuclears.  But  these  leucocytes 
take  up  only  a  relatively  small  number  of  the  bacteria  injected, 
which  develop  unrestrictedly  and  soon  become  very  numerous. 
Although  the  influx  of  the  leucocytes  is  considerable  they  never 
become  sufficient  to  give  a  purulent  appearance  to  the  exudate 
even  several  hours  after  inoculation.  As  soon  as  bacteria  become 
very  numerous  the  leucocytes  do  not  sensibly  increase  in  number. 
At  this  stage  death  is  not  very  far  distant.  A  rabbit  that  has 
received  0.1  of  a  cubic  centimeter  of  a  culture  into  the  peritoneal 
cavity  usually  dies  in  8  or  10  hours,  rarely  as  late  as  12  hours.  The 
greatest  number  of  leucocytes,  as  a  rule,  are  present  4  to  6  hours 
after  injection. 

A  short  time  before  death  the  exudate  changes  remarkably  in 
appearance.  Two  hours  before  death  it  is  only  slightly  cloudy. 
At  death  the  exudate  is  reddish  in  color  and  composed  of  serum 
which  contains  a  rather  large  number  of  scattered  red  blood  cells. 
In  this  fluid  leucocytes  are  still  present  and  they  are  frequently 
intact  in  appearance,  but  often  degenerated  and  with  no  distant 
protoplasmic  outlines.  The  leucocytes,  which  were  previously 
abundant,  are  usually  found  collected  in  masses  on  the  walls  of 
the  peritoneum,  particularly  about  the  mesentery.  The  exudate 
changes,  then,  in  composition  toward  the  end  and  becomes  a  harm- 
ful medium  for  leucocytes  and  red  blood  cells. 

Two  hours  before  death,  when  the  exudate  is  no  longer  turbid,  the 
leucocytes  present  in  the  fluid  which  refuse  to  take  up  the  strepto- 
coccus will  still  take  up  other  organisms  (for  example,  the  diphtheria 
bacillus).  Phagocytosis,  however,  under  these  conditions  is  never 
so  energetic  as  under  similar  conditions  in  the  guinea-pig.  In 
general,  it  may  be  said  that  the  exudate  in  streptococcus  peritonitis 
in  the  guinea-pig  is  always  richer  in  leucocytes  than  it  is  in  the 
rabbit. 

A  generalized  infection,  that  is,  an  invasion  of  the  blood  by  the 
streptococci,  occurs  soon  after  intraperitoneal  inoculations  in  the 
rabbit.  As  soon  as  the  bacteria  in  the  exudate  become  very 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  Ill 

numerous,  that  is,  in  4  or  5  hours  or  even  less  after  the  inoculation 
of  0.1  of  a  cubic  centimeter,  the  blood  is  invaded.  A  culture 
taken  at  this  time  from  the  heart's  blood  grows  as  luxuriantly  as 
from  the  peritoneum.  Three  hours  before  death  the  bacteria  in  the 
heart's  blood  are  still  rather  difficult  to  find  in  stained  preparations, 
but  an  hour  later  they  are  very  numerous  there  and  their  numbers 
increase  rapidly  from  this  time  on.  As  a  general  rule  a  normal 
rabbit  injected  with  streptococci,  whether  intraperitoneally  or  else- 
where, gives  an  abundant  culture  from  the  heart's  blood  at  death. 
An  examination  of  the  blood  shows  evident  changes  in  the  red 
blood  cells.  They  are  found  to  have  almost  entirely  disappeared. 
When  a  rabbit  is  autopsied  immediately  after  death,  the  heart 
contains  large  red  clots  and  serum  filled  with  diffused  hemoglobin. 
In  trying  to  express  red  blood  cells  from  this  clot  into  the  serum 
only  debris  are  obtained,  which,  when  examined  after  staining  with 
eosin,  have  no  distinct  cellular  outlines.  A  few  leucocytes,  more  or 
less  altered,  and  also  endothelial  cells  are  found. 

These  grave  alterations,  which  are  so  incompatible  with  life, 
appear  late  in  the  disease  and  only  in  the  agonal  period.  They  are 
not  to  be  found  when,  for  one  reason  or  another,  the  animal  dies 
with  few  streptococci  in  the  blood. 

When  these  alterations  do  occur  a  reddish  exudate  similar  to 
that  present  in  the  peritoneum  at  the  time  of  death  is  also  found  in 
the  pericardium  and  the  pleura.  These  late  changes  in  the  peritoneal 
exudate  (the  appearance  of  scattered  red  blood  cells  and  diffused 
hemoglobin)  are  correlative  with  changes  in  the  blood  and  appear 
only  when  such  changes  are  present. 

Animals  given  subcutaneous  injections  of  streptococci  die  with 
the  same  lesions  and  always  rapidly. 

If  streptococci  are  injected  into  the  ear  vein  of  the  rabbit,  they 
grow  in  the  blood  without  any  resistance.  For  example,  if  a  drop 
of  blood  is  taken  immediately  after  injecting  0.1  of  a  cubic  centi- 
meter and  inoculated  on  agar,  only  a  few  colonies  are  found; 
3  hours  after  injection  numerous  colonies  are  found  on  inoculating 
the  same  amount  of  blood.  After  7  hours  the  blood  gives  confluent 
colonies  of  streptococci  on  agar.  It  is  evident  that  in  the  blood, 
as  in  the  peritoneal  exudate,  there  is  no  hindrance  to  the  growth  of 
the  inoculated  micro-organisms. 


112  STUDIES  IN  IMMUNITY. 

From  these  observations,  then,  it  is  easy  to  realize  that  Mar- 
morek's  streptococcus  owes  its  extreme  virulence  for  the  rabbit, 
not  only  to  the  great  rapidity  of  its  development  in  the  body  fluids, 
but  particularly  to  its  power  to  prevent  its  own  engulfment  by 
leucocytes.  It  exercises  a  negative  chemiotactic  influence  on 
rabbit  leucocytes,  and  this  repelling  action  is  characteristic  not 
only  of  streptococci  adapted  to  the  body  fluids  by  origin  in  them, 
but  also  of  organisms  grown  on  culture  media. 

Indeed,  if  0.5  of  a  cubic  centimeter  of  a  culture  is  injected  into 
the  peritoneum  of  a  rabbit  that  has  received  6  c.c.  of  bouillon  the 
night  before  and  contains,  therefore,  numerous  leucocytes,  the  ma- 
jority of  these  bacteria  remain  free  and  soon  become  surrounded 
with  an  areola.  And  yet  under  such  conditions  the  number  of 
bacteria  injected  is  relatively  small  when  compared  with  the  large 
number  of  phagocytes.  We  know,  moreover,  that  the  phagocytes 
present  in  an  exudate  caused  by  injecting  bouillon,  are  very  ac- 
tive and  show  remarkable  phagocytic  power  for  various  bacteria. 

In  this  experiment,  however,  phagocytosis  is  not  entirely  absent; 
there  are  always  a  few  cocci  that  become  the  prey  of  cells.  If  the 
number  of  bacteria  inoculated  is  markedly  diminished,  if,  for  ex- 
ample, 0.1  of  a  cubic  centimeter  is  injected  into  the  prepared  peri- 
toneum of  such  a  rabbit,  the  number  of  engulfed  bacteria  increases; 
this  fact  indicates,  it  would  seem,  that  there  are  certain  cells  in 
this  exudate  that  are  particularly  active  and  absorb  a  certain  num- 
ber of  bacteria.  But  however  small  may  be  the  dose  inoculated, 
there  always  remain  certain  free  bacteria  whose  multiplication 
rapidly  takes  place.  A  previous  inoculation  of  bouillon  into  the 
peritoneal  cavity  increases  the  number  of  active  leucocytes  in  the 
exudate,  but  will  not  protect  the  rabbit  against  a  subsequent 
inoculation  of  streptococci  even  when  these  organisms  are  very 
few  in  number  in  proportion  to  the  phagocytes. 

This  chemiotactic  influence  is  present,  particularly,  in  young 
streptococci  and  is  present,  most  markedly,  in  organisms  under- 
going active  multiplication.  It  is  easy  to  prove  that  the  strepto- 
cocci from  a  three-  or  four-day-old  culture  are  much  less  repelling. 
If  even  a  large  dose  of  such  a  culture  (several  cubic  centimeters)  is 
injected  into  the  prepared  peritoneum  of  a  guinea-pig,  active  phago- 
cytosis occurs,  and  usually  of  the  majority  of  the  organisms 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  113 

present;  the  same  culture,  when  24  hours  old,  entirely  escapes  such 
phagocytosis.  Even  with  the  old  culture,  however,  new  organ- 
isms are  formed  in  the  exudate  after  a  certain  time  and  kill  the 
animal. 

The  fluid  in  which  cultures  have  been  grown  for  several  days 
apparently  does  not  contain  any  perceptible  amount  of  the  substance 
that  prevents  phagocytosis.  Streptococci  injected  into  the  peritoneal 
cavity  of  a  guinea-pig  prepared  by  bouillon  are  rapidly  and  more 
or  less  completely  taken  up,  depending  on  the  dose  employed.  This 
engulfing  occurs  to  just  as  marked  a  degree  if  3  c.c.  of  a  filtrate  from 
a  24-hour  culture  is  injected  with  the  bacteria.  The  negative 
chemiotactic  influence,  then,  is  a  property  which  belongs  exclusively 
to  living  streptococci.  We  should  like  to  insist  on  a  correlation 
between  a  negative  chemiotactic  influence  coming  from  the  bac- 
teria and  the  presence  of  an  areola  about  them ;  this  areola  may 
develop,  not  only  about  bacteria  in  the  peritoneal  cavity,  but  also 
under  the  skin,  in  the  blood  and  in  the  aqueous  humor. 

The  resistant  qualities  of  streptococci.  —  The  property  of  repel- 
ling leucocytes,  which  is  of  such  value  to  the  streptococcus,  is 
not  the  only  property  which  this  organism  utilizes  in  growing  in 
animal  tissues.  We  have  already  seen  that  a  fatal  relapse  may 
occur  in  guinea-pigs  even  several  days  after  an  apparent  cure. 

The  momentary  arresting  of  the  infection  and  the  apparent 
cure  are  due  to  the  intervention  of  complete  phagocytosis  and  the 
consequent  disappearance  of  free  bacteria.  These  cases,  which 
in  guinea-pigs  are  very  rare,  indicate  that  certain  streptococci  may 
be  very  resistant  and  may  remain  alive  within  phagocytes,  regain 
their  activity,  and  after  several  days  give  rise  to  a  new  culture 
that  kills  the  animal. 

This  delayed  outgrowth  of  streptococci,  after  an  apparent  -cure 
of  the  animal  lasting  some  days,  happens  in  rabbits  that  have  been 
partially  immunized  by  the  injection  of  a  certain  amount  of  pre- 
ventive serum.  The  following  experiment  shows  that  the  strepto- 
cocci in  such  a .  rabbit  may  remain  latent  for  a  long  time  in  the 
phagocytes  and  after  a  long  interval  grow  again. 

A  rabbit  was  given  6  c.c.  of  pepton  bouillon  into  the  peritoneal 
cavity  and  3  c.c.  of  antistreptococcus  serum  subcutaneously.  On 
the  following  day  6  c.c.  of  a  3-day-old  culture  of  streptococcus  was 


114  STUDIES  IN  IMMUNITY. 

inoculated  into  the  peritoneum,  which  contained  many  leucocytes. 
As  we  have  already  shown,  these  old  cultures  are  much  more  easily 
phagocyted  than  are  young  cultures.  The  exudate  examined  3 
or  4  hours  after  injection  showed  no  free  bacteria;  all  the  cocci  had 
been  taken  up  by  the  cells.  Animals  similarly  inoculated,  but 
without  serum,  usually  die  in  24  hours.  The  animal  that  has  re- 
ceived the  serum  resists  infection  and,  on  the  following  day,  the 
exudate  contains  many  leucocytes,  some  of  them  containing  more 
or  less  altered  streptococci;  there  are  no  free  bacteria.  Nor  are 
there  any  bacteria  present  the  following  day,  and  it  would  seem  as 
if  they  had  been  entirely  taken  up.  The  animal,  however,  although 
remaining  well  for  several  days,  at  the  end  of  the  week  succumbs  to 
a  generalized  streptococcus  infection.  These  instances  of  a  late 
outgrowth  of  streptococci  are  frequently  met  with  in  experiments ; 
they  occur,  irrespective  of  the  point  of  inoculation,  in  animals  that 
have  received  either  too  small  a  dose  of  serum  or  too  large  a  dose 
of  bacteria.  In  such  cases  death  may  not  occur  for  two  weeks  and, 
exceptionally,  not  even  until  the  third  week.  To  sum  up,  the  viru- 
lent streptococcus  has  two  qualities  that  render  it  dangerous,  and 
particularly  so  for  the  rabbit:  it  repels  leucocytes,  and  it  can  remain 
living  for  a  long  time  in  an  animal  that  is  apparently  cured. 

A  COMPARISON  OF  STREPTOCOCCUS  INFECTION  IN  THE  GUINEA- 
PIG  AND  IN  THE  RABBIT. 

We  must  now  compare  the  appearance  of  the  streptococcus  in- 
fection in  the  guinea-pig  and  in  the  rabbit  in  order  to  understand 
why  the  first  of  these  animals  resists  the  infection  so  much  better 
than  the  latter. 

We  have  already  shown  in  a  previous  article  that  the  normal 
serum  of  rabbits  or  of  guinea-pigs  is  a  favorable  culture  medium  for 
the  streptococcus  and  has  no  bactericidal  power  against  it. 

We  have  just  shown,  moreover,  that  streptococci  grow  very  well 
and  rapidly  in  the  peritoneal  exudate  of  infected  guinea-pigs;  the 
only  condition  necessary  for  a  uniform  development  is  that  they 
remain  free  in  the  fluid  and  protect  themselves  from  the  phagocytes 
by  their  negative  chemiotactic  power.  The  guinea-pig,  then,  has 
no  more  antiseptic  properties  in  its  fluids  than  has  the  rabbit.  But 
there  is  a  very  striking  difference  between  the  phagocytes  of  these 


STUDY  OF  ANTISTREPTOCOCCUS   SERUM.  115 

two  animals :  guinea-pig  leucocytes  are  much  less  sensitive  than  rabbit 
leucocytes  to  the  repelling  action  of  the  streptococcus.  As  much  as 
0.5  of  a  cubic  centimeter  of  a  young  culture  of  streptococcus  or 
even  more  is  rapidly  taken  up  in  the  peritoneal  cavity  of  a  guinea- 
pig  previously  injected  with  bouillon.  The  same  amount  of  strepto- 
cocci injected  into  a  rabbit  similarly  prepared  suffers  very  little 
engulfing  and  extracellular  development  occurs  rapidly. 

This  difference  in  reaction  of  the  leucocytes  explains  clearly  the 
difference  in  the  evolution  of  the  disease  in  the  two  animals. 

The  exudate  in  a  guinea-pig  given  a  fatal  dose  is  always  richer  in 
leucocytes;  the  purulent  peritonitis  which  usually  occurs  in  an 
infected  guinea-pig  is  not  found  in  an  infected  rabbit  at  autopsy. 
When  a  guinea-pig  dies  as  the  result  of  an  intraperitoneal  injection 
the  exudate  contains,  in  addition  to  a  large  number  of  leucocytes, 
numerous  streptococci.  The  blood  of  such  an  animal,  however,  con- 
tains fewer  organisms  than  does  that  of  a  rabbit  under  the  same  con- 
ditions. It  need  scarcely  be  insisted  upon  that  the  more  active  the 
phagocytic  apparatus  against  a  given  bacterium,  the  more  difficult 
does  the  invasion  of  the  blood  by  this  organism  become.  The 
extreme  alterations  in  the  blood  (destruction  of  red  blood  cells) 
found  in  the  blood  of  rabbits  and  resulting  from  an  abundant  growth 
of  the  streptococcus  is  not  found  in  the  guinea-pig  at  autopsy. 

Cases  of  streptococcus  reinfection  occurring  after  a  period  of 
apparent  cure,  although  rare  in  guinea-pigs,  are,  on  the  contrary, 
very  frequent  in  rabbits  that  have  received  a  small  dose  of  pro- 
tective serum.  What  is  the  cause  of  this  difference? 

Among  the  leucocytes  that  take  up  bacteria  there  are  always  a 
certain  number  that  die  after  having  destroyed  the  parasites  that 
they  contain;  and  so  it  is  easy  to  understand  how  such  bacteria, 
which  may  be  almost  intact,  are  liberated.  These  liberated  bacteria 
are  much  more  likely  to  be  taken  up  again  in  a  guinea-pig  than  in  a 
rabbit,  since  the  guinea-pig  leucocytes  are  more  actively  phagocytic  for 
the  streptococcus.  It  may  be,  too,  that  guinea-pig  leucocytes  have 
a  greater  destructive  power  for  the  streptococcus  than  rabbit  leu- 
cocytes. This  leads  us  to  va  consideration  of  the  alterations  in 
streptococci  taken  up  by  cells  in  the  two  animals  we  are  studying. 
In  both  of  these  animals  the  leucocytes  cause  destructive  altera- 
tions in  at  least  some  of  the  phagocyted  bacteria. 


116  STUDIES  IN  IMMUNITY. 

When  streptococci  have  remained  for  several  hours  in  the  pro- 
toplasm of  the  phagocyte  some  of  them  take  acid  dyes  in  preference 
to  basic  dyes;  in  a  counter  stain  with  eosin  and  methylene  blue 
such  bacteria  take  a  more  or  less  red  stain.  This  phenomenon 
occurs  both  in  the  rabbit  and  in  the  guinea-pig,  but  in  the  latter  is 
more  evident  on  account  of  the  greater  extent  of  the  phagocytosis. 

II. 
ANTISTREPTOCOCCUS   SERUM. 

Marmorek,  in  an  article  published  in  July,  1895,  in  the  Pasteur 
Annals,  has  described  his  method  of  obtaining  this  serum  by  im- 
munizing animals  against  the  streptococcus. 

We  shall  not  reiterate  his  methods.  It  may  be  noted  simply  that 
the  serum  we  have  used  was  from  animals  that  had  been  under 
immunization  for  about  a  year.  One  of  these,  the  serum  of  which 
was  remarkably  active,  had  received  twenty- three  injections  of 
a  very  virulent  24-hour  culture.  The  total  amount  of  culture 
injected  during  this  time  was  3800  c.c. 

The  preventive  activity  of  these  sera  is  most  evident.  When 
injected  into  a  rabbit  before  inoculation  of  bacteria,  they  permit  the 
animal  to  withstand  many  times  the  minimal  fatal  dose  of  strepto- 
coccus. 

The  amount  of  bacteria  that  can  be  safely  injected  in  animals 
immunized  by  serum  varies  a  great  deal  according  to  the  region 
in  which  the  injection  is  given.  Animals  that  have  received 
serum  do  not  tolerate  intraperitoneal  injections  of  streptococci 
nearly  as  well  as  they  do  subcutaneous  injections.  Intravenous  or 
intraocular  injections  are  also  much  more  dangerous,  even  in 
animals  that  have  received  a  very  highly  active  serum.  In  deter- 
mining the  preventive  value  of  a  given  serum  the  portal  of  entry 
chosen  for  inoculation  of  the  culture  should  therefore  be  indicated. 
For  this  reason  we  shall  frequently  mention  the  doses  of  serum  and 
culture  used  in  our  experiments. 

A  few  figures  from  our  notebook  may  be  given  at  once  as  show- 
ing the  result  of  injecting  a  culture  subcutaneously  in  passively 
immunized  animals. 

An  animal  that  has  received  10  c.c.  of  serum  subcutaneously  can 


STUDY  OF   ANTISTREPTOCOCCUS   SERUM.  117 

be  given  0.5  of  a  cubic  centimeter  of  culture  subcutaneously  the 
next  day  without  any  effect.  A  control  given  0.001  of  a  cubic 
centimeter  of  culture  dies.  The  rabbit  that  receives  serum  remains 
perfectly  well,  although  it  has  received  at  least  500  times  the  fatal 
dose  of  streptococci. 

A  smaller  dose  of  serum  (5  c.c.)  protected  a  rabbit  against  0.001 
of  a  cubic  centimeter  of  a  virulent  culture,  while  the  control  that 
had  received  one-tenth  of  this  amount  (that  is  0.0001)  died.  We 
offer  these  figures,  not  as  a  systematic  tabulation  of  the  preventive 
power  of  horse  serum  but  as  results  that  occurred  regularly  during 
our  experiments.* 

A  suitable  dose  of  preventive  serum,  as  in  the  case  just  men- 
tioned, protects  rabbits  against  the  inoculation  of  streptococci. 
If  too  small  doses  in  proportion  to  the  number  of  organisms  in- 
jected are  given,  the  animals  may  simply  resist  longer  than  the 
controls  or  show  an  apparent  cure  lasting  long  after  any  original 
manifestation  of  the  disease,  but  eventually  succeeded  by  a  rapid 
reinfection  with  the  streptococcus. 

These  conditions  will  be  considered  later.  For  the  moment  we 
shall  consider  briefly  the  effect  of  the  sera  on  bacteria  in  vitro. 

1.  ACTION  OF  THE  SERUM  IN  VITRO. 

Marmorek's  preventive  serum  is  from  a  horse  immunized  against 
the  streptococcus.  The  serum  has  no  bactericidal  power  for  the 
streptococcus.  Bacteria  inoculated  in  it  do  not,  to  be  sure,  grow 
very  rapidly  or  abundantly,  but  they  grow  quite  as  well  in  the 
immune  serum  as  they  do  in  normal  horse  serum. 

Preventive  serum  mixed  with  fresh  normal  rabbit  serum  has  also 
no  inhibiting  power  on  the  growth  of  the  organism.  Streptococci 
grow  equally  well  in  a  mixture  composed  of  1.5  c.c.  of  normal  rabbit 
serum  and  5  c.c.  of  preventive  serum,  or  in  a  mixture  in  like  pro- 

*  Petruschky,  in  two  recently  published  articles  (Zeitschrif t  fur  Hygiene) ,  as  a 
result  of  his  experiments  concludes  that  Marmorek's  serum  does  not  in  the  least 
protect  rabbits  against  streptococcus  infection.  Treated  rabbits  showed  no 
appreciable  difference  over  the  controls  according  to  this  author.  It  is  evident 
that  Petruschky  has  experimented  with  a  serum  that  was  spoiled  in  some  manner. 
The  efficacy  of  Marmorek's  serum  may  be  shown  by  the  most  elementary  experi- 
mentation, and  consequently  the  experiments  and  conclusions  of  Petruschky 
need  not  concern  us  as  they  do  not  deal  with  antistreptococcus  serum  as  obtained 
at  the  Pasteur  Institute. 


118  STUDIES  IN  IMMUNITY. 

portions  with  normal  horse  serum  instead  of  preventive  serum. 
Streptococci  grown  in  these  media,  however,  show  certain  peculiari- 
ties that  may  be  noted. 

In  both  these  mixtures  of  normal  rabbit  serum  and  either  normal 
or  immune  horse  serum  the  growth  begins  rapidly  if  the  cultures 
inoculated  are  young.  An  equal  and  considerable  growth  is  evident 
three  hours  after  inoculation.  In  the  tube  containing  the  normal 
horse  serum  the  streptococci  are  in  numerous  short  chains;  in  the 
other,  containing  preventive  serum,  they  form  much  fewer  and 
longer  chains  and  these  chains  are  frequently  coiled.  There  is  a 
difference  then  in  the  arrangement  of  the  cocci,  although  the  total 
number  is  about  the  same  in  either  medium. 

After  5J  hours  these  same  characteristics  are  still  present.  Later 
on  (19  hours  after  inoculation)  the  chains  become  longer  in  the  tube 
containing  normal  serum,  so  that  the  difference  in  length  of  chains 
between  the  two  tubes  is  less  manifest.  There  is  still  no  difference 
to  be  noted  in  the  richness  of  the  culture.  We  have  found  no 
retarding  property  on  growing  streptococci  in  antistreptococcus 
horse  serum  any  more  than  did  Denys  and  Marchand.* 

If  in  such  an  experiment  a  less  virulent  streptococcus  is  used 
(one  that  kills  a  rabbit  in  a  dose  of  0.25  of  a  cubic  centimeter  in- 
travenously), similar  results  are  obtained;  with  such  an  organism, 
however,  the  difference  in  the  length  of  the  chains  is  much  less. 

The  injection  of  10  c.c.  of  preventive  serum  in  a  normal  rabbit 
does  not  endow  this  animal's  serum  with  any  bactericidal  power. 
The  serum  25  hours  after  injection  is  just  as  good  as  a  culture 
medium  for  streptococcus  as  before  the  injection  of  antistrepto- 
coccus serum.  In  both  of  them  it  grows  rapidly  and  well. 

Antistreptococcus  serum  has  a  slight  but  distinct  agglutinating 
property.  We  demonstrated  in  1895  that  a  trace  of  a  preventive 
cholera  serum,  when  introduced  in  a  fluid  containing  cholera 
vibrios  in  suspension,  produces  in  a  very  short  time  their  immobiliza- 
tion, and  clumps  them  into  distinct  masses  that  stand  out  as  white 
points  in  a  clarified  fluid.  This  is  the  invariable  effect  of  this  pre- 
ventive serum.  The  following  year  Gruber  and  Durham  noted  a 
similar  fact,  not  only  with  vibrios,  but  also  with  B.  typhosus  and 

*  Denys  and  Marchand,  Immunity  conferee  au  lapin  par  1'injection  de  se'rum 
anti-streptococcique  de  cheval.  Bull.  Academic  royale  de  Belgique,  1896. 


STUDY   OF  ANTISTREPTOCOCCUS  SERUM.  119 

B.  coli,  all  of  which  are  found  to  clump  when  placed  in  contact 
with  a  specific  serum. 

Although  the  agglutination  of  the  streptococcus  by  its  preventive 
serum  is  generally  distinct,  it  is  never  very  marked;  to  cause  it, 
large  quantities  of  serum  are  necessary,  at  least  one-third  the 
volume  of  the  culture  fluid.  Not  all  of  the  chains  of  the  strepto- 
cocci are  affected;  some  of  them  remain  separate.  Cultures  of 
streptococci  grown  in  preventive  serum  or  in  a  fluid  containing  a 
certain  amount  of  it  (for  example,  equal  parts  of  bouillon  and 
immune  serum)  show  only  a  slight  grouping  of  the  organisms  at 
best.  In  short,  preventive  serum  causes  no  profound  alteration  in 
the  streptococcus.  The  growth  of  the  organism  is  not  sensibly 
diminished  and  its  morphology  remains  the  same,  with  the  excep- 
tion of  certain  variations  in  the  length  of  the  chains.  And  even 
the  agglutinating  property  that  has  been  found  by  recent  studies 
to  be  present  in  numerous  immune  sera  is  only  slightly  developed  in 
antistreptococcus  serum. 

We  must  consider  whether  a  preventive  serum  with  so  slight  an 
effect  on  the  morphology  and  development  of  the  streptococcus 
does  not  have  some  weakening  influence  on  its  virulence.  Cul- 
tures of  streptococci  grown  in  a  mixture  of  equal  parts  of  preven- 
tive serum  and  pepton  bouillon  retain  a  very  great  virulence,  as 
may  be  determined  by  comparing  them  with  cultures  made  on 
bouillon  plus  normal  horse  serum.  After  24  hours'  growth  in  an 
incubator  the  turbid  culture  fluids  are  filtered  through  filter  paper. 
There  remain  enough  bacteria  on  the  filter  paper  and  they  may  be 
washed  and  freed  of  serum.  A  small  amount  of  these  bacteria  is 
taken  with  sterile  forceps  and  suspended  in  a  few  cubic  centimeters 
of  salt  solution.  Two  emulsions  are  thus  obtained,  both  slightly 
and  as  near  as  possible  equally  turbid:  one  containing  bacteria 
cultivated  in  normal  serum  and  the  other  bacteria  cultivated  in 
preventive  serum.  Both  these  fluids  are  extremely  virulent,  al- 
though they  contain  few  bacteria;  0.0005  of  a  cubic  centimeter  of 
either  one  kills  a  rabbit  in  24  hours.  In  other  words,  there  has 
been  no  detectable  attentuation  by  growing  in  preventive  serum. 

And,  what  is  more,  the  whole  culture  containing  preventive  serum 
and  bacteria  is  also  very  dangerous  for  rabbits,  killing  them  as 
rapidly  as  controls,  that  is,  in  24  hours. 


120  STUDIES  IN   IMMUNITY. 

The  latter  result  may  seem  astonishing.  One  might  expect 
rabbits  that  have  received  the  whole  culture  to  receive  some  benefit 
from  the  serum  in  the  fluid  and  to  be  enabled  to  resist  the  invasion 
of  the  bacteria.  But  it  must  be  noted  that  this  culture  contains  a 
considerable  number  of  bacteria,  and  experiment  shows  us  that 
1  c.c.  of  pure  serum  fails  to  protect  animals  against  a  much  smaller 
dose  of  bacteria  than  is  present  in  a  cubic  centimeter  of  such  a 
culture.  It  is  not  surprising,  then,  that  the  culture  is  virulent,  for 
it  contains  too  many  bacteria  in  proportion  to  the  serum. 

This  experiment  that  we  have  just  mentioned*  'simply  shows 
that  streptococci  grown  in  preventive  serum  show  no  deterioration 
that  decreases  their  virulence  in  subsequent  generations.  Does 
it  show,  however,  that  preventive  serum  when  injected  into  an 
animal  before  infection  is  quite  incapable  of  any  enfeebling  effect 
on  the  subsequently  injected  steptococci?  By  no  means,  for  we 
may  imagine  that  a  serum  incapable  of  modifying  bacteria  in  vitro 
may,  when  in  the  tissues,  affect  them,  owing  to  the  additional  or 
combined  effect  of  certain  adjuvant  factors  furnished  by  the  animal 
body.  Have  we  not  seen,  for  example,  that  cholera  serum  which, 
when  kept  for  some  time  or  heated,  is  incapable  of  causing  granular 
transformation  in  vitro  can  bring  about  this  modification  when  in- 
jected into  an  animal?  We  make  these  reservations  on  a  priori 
grounds  without  considering  for  the  moment  whether  or  not  they 
are  justified  by  experimental  facts. 

The  fluids  hitherto  examined,  that  is,  normal  rabbit  serum,  and 
preventive  serum  either  pure  or  mixed  with  bouillon  or  rabbit 
serum,  have  no  bactericidal  effect  on  the  streptococcus.  There 
is  a  body  fluid,  however,  which  shows  an  evident  destructive  effect 
on  this  micro-organism.  It  is  the  clear  fluid  separated  by  centrif- 
ugalizing  an  exudate  rich  in  leucocytes.  The  streptococcus  grows 
with  great  difficulty  in  this  fluid  and,  as  Denys  and  Leclef  have 
already  noted,  a  destruction  of  bacteria  ensues  even  when  a  large 
amount  of  culture  is  inoculated. f  If  a  drop  of  a  young  culture  is 

*  This  experiment  is  similar  to  those  of  Metchnioff  (1892),  Issaeff,  and 
Sanarelli  (1893)  with  the  bacteria  of  hog-cholera,  pneumonia  and  the  vibrio 
Metchnikovi. 

f  Denys  et  Leclef,  Sur  I'lmmunite"  du  lapin  vaccine"  centre  le  streptocoque.  La 
Cellule,  1895. 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  121 

inoculated  in  such  a  fluid,  no  development  takes  place  and  the  next 
day  the  fluid  is  found  to  be  sterile. 

This  serous  fluid  loses  its  bactericidal  power  when  heated  for  a 
few  moments  to  60  degrees,  and  in  such  heated  fluid  the  strepto- 
coccus grows  in  long  chains  of  rather  small  organisms.  As  the 
fluid  part  of  rich  leucocytic  exudate  is  bactericidal  in  vitro,  the 
question  arises  as  to  whether  it  is  as  much  so  in  the  animal  body. 
Experiment  gives  a  negative  reply  to  this  question.  When  strepto- 
cocci are  injected  into  the  rabbit  peritoneal  cavity  containing  many 
leucocytes  the  organisms  are  not  engulfed,  but  develop  well.  If 
a  little  of  this  exudate  is  removed  when  the  streptococci  are  not  yet 
very  numerous,  and  placed  in  the  incubator,  the  growth  stops;  at 
times  the  fluid  becomes  quite  sterile  after  2  or  3  days  and  at 
other  times  the  organism  finally  grows  out  again  after  a  delay  of 
24  hours  or  so. 

Experiments  performed  in  vitro  with  leucocytic  exudates  can- 
not be  accepted  offhand  without  many  reservations  as  necessarily 
corresponding  to  the  conditions  of  similar  experiments  in  the 
animal  body. 

2.   THE  ACTION  OF  THE  SERUM  IN  THE  ANIMAL  BODY. 
INTRAPERITONEAL  INJECTION. 

Let  us  now  consider  the  phenomenon  which  takes  place  in  rabbits 
that  have  received  a  preventive  injection  of  serum  and  are  sub- 
sequently inoculated  with  the  steptococcus.  We  shall  consider 
first  an  instance  in  which  the  serum  is  injected  subcutaneously 
24  hours  before  the  culture  is  injected  intraperitoneally. 

The  study  of  the  struggle  between  the  cells  .and  the  bacterial 
culture  in  the  peritoneal  cavity  is  extremely  instructive  as,  by 
extracting  at  intervals  a  little  peritoneal  exudate  with  a  capillary 
tube,  the  course  of  the  infection  may  be  followed  step  by  step. 

The  exudate  is  homogeneous,  that  is  to  say,  throughout  the 
peritoneal  cavity  it  has  an  identical  constitution  in  so  far  as  the 
number,  quality,  and  appearance  of  cells  and  bacteria  is  concerned. 
The  subcutaneous  exudate,  on  the  contrary,  frequently  differs  even 
in  adjacent  regions,  either  in  the  amount  of  fluid  or  in  the  number 
and  condition  of  cells  and  bacteria. 

It  is  possible  to  produce  artificial  variations  in  the  number  of 


122  STUDIES  IN  IMMUNITY. 

cells  present  in  the  peritoneal  exudate  before  injecting  bacteria. 
By  means  of  a  simple  injection  of  bouillon  a  great  many  active 
phagocytes  may  be  made  to  participate  at  the  very  beginning  of 
the  infection.  This  method  is  very  useful  in  studying  the  course 
of  an  infection  and  we  have  frequently  employed  it. 

In  order  to  understand  the  phenomena  of  a  peritoneal  infection 
in  passively  immunized  animals  we  must  consider  a  fact  evidenced 
on  simultaneous  injection  of  streptococci  into  a  normal  and  into 
an  immunized  rabbit.  A  virulent  streptococcus  inoculated  into 
the  circulation  of  a  normal  rabbit  in  a  dose  of  0.1  to  0.25  of  a  cubic 
centimeter  of  culture  develops  so  well  as  to  give  an  abundant  culture 
from  the  blood  in  seven  or  eight  hours.  The  same  dose  of  culture 
injected  intravenously  into  a  rabbit  that  has  received  serum  sub- 
cutaneously  is  markedly  inhibited  in  growth.  A  drop  of  blood 
taken  from  any  part  of  the  body  immediately  after  injection  and 
inoculated  on  agar  gives  rise  to  a  few  colonies.  Streptococci  may 
still  be  found  in  the  blood  five  to  seven  hours  later  or  even  on  the 
following  day,  but  their  number  is  not  increased.  Such  a  rabbit, 
however,  succumbs  in  two,  three,  or  four  days,  but  at  autopsy  many 
less  bacteria  are  found  in  the  blood  than  are  present  in  the  rabbit 
that  has  received  no  serum.  More  bacteria,  it  is  true,  are  found  in 
the  liver,  spleen,  bone  marrow,  and  particularly  in  the  lung;  some 
of  them  free  and  others  within  leucocytes. 

In  this  connection  we  may  emphasize  the  fact  that,  as  a  general 
rule,  in  rabbits  that  have  received  an  injection  of  serum  and  an 
excessive  dose  of  bacteria  and  have  survived  the  controls  only  two 
or  three  days,  there  are  never  so  many  bacteria  present  in  the  heart's 
blood  as  there  are  in  control  animals.  The  contrast  is  very  marked 
from  whatever  region  the  cultures  are  made. 

The  only  exceptions  to  this  rule  are  those  instances  where  rabbits 
that  have  apparently  been  cured,  after  a  certain  time  have  a  fatal 
relapse,  under  which  conditions  the  blood  may  contain  nearly  as 
many  bacteria  as  a  rabbit  infected  without  serum. 

The  peritoneal  infection  in  normal  rabbits  is  accompanied  by 
a  rapid  invasion  of  the  blood  by  the  micro-organism;  the  increase 
of  streptococci  in  the  blood  is  so  marked  that  it,  and  not  the 
peritonitis,  must  be  regarded  as  the  immediate  cause  of  death. 
Therefore,  since  there  is  less  growth  of  bacteria  in  the  blood  of 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  123 

the  vaccinated  animal,  a  peritoneal  infection  in  such  an  animal 
may  last  much  longer  than  in  a  control,  without  leading  to  a 
septicemia.  This  is  the  point  of  importance  to  us  for  the  moment. 

A.  Young  cultures.  —  What  happens  to  virulent  streptococci 
in  a  young,  24-hour  culture  when  injected  into  the  peritoneum  of 
a  rabbit  containing  many  leucocytes  owing  to  a  previous  injection 
of  10  c.c.  of  serum  subcutaneously  and  6  c.c.  of  bouillon  intraperi- 
toneally?  There  are  two  distinct  conditions  to  be  considered. 

First:  —  The  number  of  bacteria  injected  may  be  very  few,  less 
than  0.5  of  a  cubic  centimeter  of  culture;  let  us  take,  for  example, 
0.1  of  a  cubic  centimeter.  Under  these  conditions  the  number  of 
bacteria  in  the  peritoneal  cavity  is  relatively  small  in  proportion 
to  the  number  of  cells,  so  that  they  are  frequently  difficult  to  find 
in  stained  preparations.  Under  these  conditions  the  culture  is 
rapidly  and,  as  far  as  may  be  estimated,  completely  engulfed.  For 
example,  after  an  hour  or  two  no  free  cocci  are  found ;  when  stained 
with  boracic  carmin  followed  by  Gram  they  are  seen  to  have  be- 
come the  prey  of  cells.  As  a  result,  no  extracellular  development 
of  bacteria  takes  place  under  these  conditions  and  the  animal  gets  well. 
In  a  control  animal  that  has  received  an  intraperitoneal  injection 
of  bouillon  but  no  protective  serum  a  very  distinct  phagocytosis  is 
generally  found  if  small  doses  of  bacteria  are  injected;  free  bacteria 
are  only  exceptionally  found  in  such  preparations.  A  multiplica- 
tion, nevertheless,  takes  place,  and  within  three  or  four  hours 
numerous  cocci  with  an  intense  negative  chemiotactic  influence 
are  found.  Such  an  animal  usually  dies  within  twelve  hours 
with  the  ordinary  symptoms  occurring  in  normal  animals  as 
already  described. 

Second :  —  The  results  are  quite  different  if  instead  of  a  very  small 
amount  of  culture,  that  is  0.1  of  a  cubic  centimeter,  a  dose  of  0.5  of  a 
cubic  centimeter  or  slightly  more  is  given.  Under  these  conditions* 

*  We  may  mention  that  when  0.5  of  a  cubic  centimeter  of  a  culture  is  injected 
into  a  peritoneum  prepared  by  bouillon  the  number  of  streptococci  at  first  in  the 
exudate  is  very  small  in  proportion  to  the  number  of  cells.  Notwithstanding  this 
fact  reproduction  occurs  and  as  we  shall  see  presently  the  animal  is  saved  only  by 
a  delayed  phagocytosis. 

Denys  and  Leclef  (La  Cellule,  1895)  made  a  mixture  of  normal  rabbit  leucocytes 
and  preventive  serum  in  vitro.  When  they  injected  this  mixture  with  the  strep- 
tococcus they  found  there  was  a  distinct  inhibition  in  the  growth  of  their  culture 
owing  to  an  abundant  initial  phagocytosis,  although  the  organism  grows  rapidly 


124  STUDIES   IN   IMMUNITY. 

the  engulfing  that  takes  place  in  the  first  few  hours  is  only  partial. 
As  in  the  previous  experiment  streptococci  are  found  within  appar- 
ently vigorous  cells,  but  since  the  number  of  streptococci  injected 
is  too  great,  many  of  them  remain  free  and  develop.  In  these 
doses  there  is  not  so  much  difference  as  regards  phagocytes  between. 
a  rabbit  treated  by  serum  and  a  normal  rabbit.  The  streptococci 
increase  without  any  notable  retarding  effect  on  their  development. 
Eight  or  ten  hours  after  injection  the  organisms  in  the  vaccinated 
animals  are  surrounded  by  an  areola  and  are  growing  in  the  midst 
of  leucocytes  while  the  control  is  nearing  death.  The  animal  re- 
covers, however,  although  the  bacteria  are  always  extremely  numer- 
ous and  phagocytosis  is  either  insignificant  or  absent. 

The  appearance  of  the  exudate  changes,  however.  It  becomes 
thicker  and  thicker  and  more  concentrated  until  it  is  almost  white 
and  its  proportion  of  leucocytes  great.  This  condition  lasts  for  a 
longer  or  shorter  period.  When  the  thickened  exudate  comes  to  re- 
semble a  homogeneous  white  pus,  say  20  hours  after  injection,  phago- 
cytosis suddenly  appears.  Within  a  few  hours  later,  3  or  4  at  the 
most,  all  the  streptococci  that  were  swarming  outside  the  cells  are  cap- 
lured  by  the  phagocytes.  A  great  majority  of  the  cells  contain  cocci 
and  often  in  numbers.  This  complete  engulfing  is  followed  by 
either  a  final  or  a  temporary  cure;  if  the  number  of  bacteria  was  too 
great  a  relapse  may  occur  2  or  3  days  later  although  the  phago- 
cytosis seemed  complete. 

This  delayed  phagocytosis  may  be  only  partial  if  the  culture  has 
developed  too  extensively.  Under  such  conditions  the  animal 
simply  lives  longer  than  the  controls.  Complete  phagocytosis  is  the 
essential  condition  for  a  cure. 

Conditions  necessary  for  the  occurrence  of  a  delayed  phagocytosis: 

Delayed  phagocytosis  may  occur  in  animals  that  have  received 
sufficient  serum  whether  subcutaneously  or  intraperitoneally.  It 
is  most  conveniently  studied  in  a  rabbit  prepared  by  an  injection 
of  bouillon  and  thereby  rendered  more  resistant  to  peritoneal  in- 
fection. Under  these  conditions  it  may  occur  even  with  an  amount 

in  a  mixture  of  serum  and  normal  rabbit  leucocytes  without  the  preventive  serum. 
We  have  obtained  different  results  under  such  conditions.  We  found  at  the 
beginning  a  certain  degree  of  phagocytosis  whether  the  preventive  serum  was 
added  or  not.  Subsequent  development  took  place  in  both  mixtures. 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  125 

of  bacteria  relatively  large  in  proportion  to  the  amount  of  serum 
employed  (for  example,  a  rabbit  may  be  given  6  c.c.  of  bouillon 
intraperitoneally  and  the  next  day  1  c.c.  of  culture  plus  5  c.c.  of 
serum;  a  delayed  and  complete  phagocytosis  occurs  about  25  hours 
later). 

Delayed  phagocytosis  may  also  occur  in  animals  that  have  simply 
received  serum  subcutaneously  without  any  preparation  of  the 
peritoneum.  But  under  these  conditions  the  dose  of  bacteria  in- 
jected must  be  considerably  less.  In  rabbits  protected  by  serum 
without  previous  intraperitoneal  preparation  by  bouillon,  an  intra- 
peritoneal  injection  is  much  more  dangerous  than  a  subcutaneous 
one  and  more  harmful  still  if  the  culture  used  is  young  and  rapidly 
growing.  Even  in  a  vaccinated  animal  the  inoculated  strepto- 
cocci find  a  very  suitable  culture  medium  in  the  limpid  exudate;* 
when  leucocytes  finally  come  up  in  considerable  numbers  they 
meet  with  a  large  number  of  bacteria. 

In  rabbits  immunized  by  serum,  in  whose  peritoneal  cavity  a 
delayed  phagocytosis  occurs,  the  number  of  phagocytes  gradually 
becomes  considerable.  In  the  first  hours,  however,  this  influx  of 
leucocytes  is  no  more  marked  than  in  normal  rabbits. 

Intraperitoneal  injection  of  filtered  culture  fluid  causes  an  abun- 
dant influx  of  leucocytes  both  in  normal  and  in  treated  rabbits. 
When  week-old  cultures  are  used  or  dead  or  attenuated  cocci, 
similar  results  occur  in  a  normal  rabbit. 

B.  Old  cultures. — Part  of  the  danger  from  injection  of  young 
cultures  lies  in  the  extreme  rapidity  of  their  increase  before  the 
phagocytes  arrive.  If  streptococci  that  do  not  show  active  repro- 
duction for  several  hours  are  used,  a  delayed  phagocytosis  occurs 
much  more  readily  even  if  the  serum  is  injected  after  the  bacteria. 
A  brief  resume  of  such  an  experiment  follows:  Two  normal  rab- 
bits received  each  an  intraperitoneal  injection  of  8  c.c.  of  a  four- 
day-old  culture  on  ascites  bouillon.  Six  hours  later  the  phagocytes 
had  become  very  numerous  and  the  bacteria  injected  were  engulfed 
(old  streptococci,  as  we  have  already  seen,  are  easily  phagocyted). 

*  We  have  never  found  a  disappearance  of  bacteria,  as  noted  by  Denys  and 
Leclef  following  inoculation  of  streptococcus  into  the  pleura  of  vaccinated 
rabbits,  after  injecting  a  small  dose  of  streptococcus  intraperitoneally  in  a  treated 
rabbit. 


126  STUDIES  IN  IMMUNITY. 

No  growth  had  as  yet  occurred  and  no  free  bacteria  were  seen. 
At  ihis  stage  one  of  the  rabbits  was  given  3  c.c.  of  preventive  serum 
intraperitoneally  and  5  c.c.  subcutaneously. 

The  other  rabbit  that  received  no  serum  died  of  a  generalized 
infection  in  18  hours,  the  delay  being  due  to  the  slow  growth  of  the 
culture.  Twenty-four  hours  after  beginning  the  experiment  the 
peritoneal  cavity  of  the  rabbit  that  had  received  serum  contained 
numerous  extracellular  bacteria  scattered  in  the  midst  of  abun- 
dant leucocytes.  Growth,  then,  had  taken  place.  But  a  few  hours 
later  the  phagocytic  crisis  occurred  and  all  the  free  bacteria  were 
engulfed. 

In  this  experiment  an  extracellular  growth  occurred  and  was 
followed  by  a  delayed  phagocytosis.  And  yet  the  serum  had  been 
given  some  time  after  the  injection  of  the  culture.  When  the  serum 
is  injected  the  day  before,  and  old  cultures  are  used  it  is  generally 
found  that  a  generalized  phagocytosis  occurs  and  is  not  followed  by 
the  appearance  of  extracellular  bacteria. 

Engulfing  may  also  take  place  in  a  normal  rabbit  and  often  no 
invasion  of  the  bacteria  is  noted  for  a  number  of  hours ;  the  strepto- 
cocci have  remained  in  a  latent  condition.  But  the  growth  soon 
starts  up  again  and  new  cocci  are  developed  and  invade  the  exudate. 
Do  these  new  organisms  come  from  the  few  streptococci  that  have 
remained  free  in  spite  of  the  generalized  phagocytosis?  Or  are 
they  derived  from  bacteria  that  were  taken  up,  but  have  resisted 
the  phagocytes?  Either  explanation  is  possible.  We  have  already 
seen  that  the  streptococci  may  remain  alive  for  a  long  time  in  the 
animal  body  even  after  they  have  been  taken  up  by  cells. 

Rabbits  treated  with  preventive  serum  may  resist  an  infection 
with  very  large  amounts  of  an  old  culture.*  In  a  general  way 
these  are  the  phenomena  in  rabbits  vaccinated  with  serum  and 
subsequently  inoculated  intraperitoneally  with  the  streptococcus. 
We  must  now  consider  delayed  phagocytosis  in  some  detail. 

*  The  activity  of  several-day-old  cultures  naturally  depends  much  on  the  nature 
of  the  culture  medium.  When  this  medium  is  better  than  usual  the  streptococci 
remain  "younger"  for  a  longer  time,  if  this  expression  may  be  permitted;  in  other 
words,  they  retain  better  their  property  of  growing  rapidly  on  a  new  soil. 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  127 

3.    DELAYED  PHAGOCYTOSIS  OR  PHAGOCYTIC  CRISIS, 

The  phagocytic  crisis  may  be  readily  studied  in  the  peritoneum 
of  rabbits  treated  with  serum.  It  occurs  suddenly  on  top  of  an 
extracellular  and  usually  abundant  development  of  bacteria;  it 
occurs  only  in  an  exudate  that  has  become  thick  and  purulent 
in  appearance;  other  things  being  equal,  it  takes  place  more  rapidly 
and  certainly  in  rabbits  prepared  by  bouillon  than  in  those  with  a 
normal  peritoneum  at  the  time  of  injection.  The  preparation  of 
the  peritoneum  allows  this  crisis  to  occur  more  readily  owing  to  the 
extreme  abundance  of  leucocytes  in  the  exudate. 

The  phagocytic  crisis  may  be  complete  or  incomplete  and  its 
time  of  occurrence  varies  according  to  the  gravity  of  the  case.  If 
the  crisis  is  incomplete  or  too  much  delayed  the  animal  simply 
survives  longer  than  the  controls.  It  frequently  occurs  after 
20  hours  and  even  later  when  incomplete. 

If  the  number  of  bacteria  developed  is  too  great  or  the  dose  of 
serum  too  small  the  animal  may  die  before  phagocytosis  is  com- 
plete; under  such  conditions  the  free  bacteria  at  the  time  of  death 
are  restricted  in  number. 

Death  may  also  occur  following  a  partial  phagocytosis  succeeded 
by  a  new  growth  of  new  streptococci  that,  owing  to  their  adaptation, 
are  again  able  to  invade  the  exudate. 

Such  an  instance  of  delayed  and  partial  phagocytosis  we  may  now 
consider  in  detail;  a  rabbit  received  6  c.c.  of  bouillon  intraperiton- 
eally  and  was  given  an  injection  the  next  day,  also  intraperitoneally, 
of  1  c.c.  of  a  streptococcus  culture  to  which  was  added  5  c.c.  of 
preventive  serum.*  In  accordance  with  the  appearance  of  the 
bacteria  and  cells  the  process  of  infection  may  be  divided  into  four 


First  stage  or  stage  of  free  development.  —  Following  the  injection 
a  very  restricted  number  of  bacteria  are  engulfed  by  the  relatively 
numerous  cells.  The  growth  of  the  organisms  takes  place  actively. 
A  preparation  made  10 J  hours  after  injection  shows  a  complete 
absence  of  phagocytosis;  the  leucocytes  are  very  numerous  however. 

*  The  control  received  0.1  of  a  cubic  centimeter  of  culture  and  5  c.c.  of  normal 
horse  serum  and  died  eleven  hours  later  with  the  usual  findings  at  autopsy. 


128  STUDIES  IN  IMMUNITY. 

The  bacteria  are  extremely  numerous,  normal  as  regards  size  and 
color  reaction,  and  are  present  in  short  chains  or  as  diplococci. 

Second  stage  or  the  stage  of  incomplete  phagocytosis. —  Twenty-two 
hours  after  injection  the  number  of  bacteria  is  not  very  much 
greater  than  at  ten  hours  and  phagocytosis  is  still  incomplete 
although  leucocytes  are  numerous  and  the  exudate  less  fluid.  The 
appearance  of  the  bacteria  in  the  fluid,  however,  remains  the  same. 
They  are,  as  a  general  thing,  distinctly  smaller  and  stain  more  faintly 
with  methylene  blue.  A  large  number  of  very  small  diplococci 
are  also  found.  There  are  to  be  noted  also  short  chains  composed 
of  very  small  cocci  which  are  frequently  compressed  or  unequally 
spaced  and  often  irregularly  colored.  More  rarely  still  indistinct 
chains,  containing  only  a  few  stained  cocci,  appear. 

There  are  found  scattered  throughout  the  exudate  well  stained 
normal  chains  with  individual  organisms  as  large  or  larger  than 
usual. 

There  is  a  very  distinct  contrast  between  these  normal  or  nearly 
normal  forms  and  the  bacteria  with  the  peculiar  characteristics  just 
mentioned.  In  other  words  the  streptococci,  instead  of  being  uni- 
form in  appearance,  show  distinct  variations. 

Third  stage  or  phagocytic  stage.  —  Six  hours  later  (that  is  28  hours 
after  injection)  there  is  generalized  phagocytosis. 

The  exudate  has  gradually  become  thicker,  and  contains  a  large 
number  of  leucocytes  of  the  polynuclear  type  and  some  mononu- 
clear  cells  of  variable  sizes.  It  is  to  be  noted  that  the  mononuclears, 
particularly  the  large  macrophages,  have  distinctly  outstripped  the 
microphages  in  phagocytic  activity.  Many  of  the  microphages  are 
empty,  whereas  the  mononuclear  cells  have  taken  up  considerable 
numbers  of  bacteria.  On  staining  with  Kiirine's  blue  it  is  found 
that  the  phagocyted  bacteria  are  generally  the  small  ones  taking 
the  faint  stain  and  showing  the  peculiarities  just  mentioned.  On 
account  of  their  smallness  and  their  poor  staining  reaction  it  is 
frequently  difficult  to  detect  them  inside  the  phagocytes  when 
staining  with  methylene  blue.  This  latter  remark  is  applicable 
to  all  instances  of  partial  or  total  delayed  phagocytosis;  some  cells, 
to  be  sure,  contain  cocci  of  normal  appearance  that  are  easily 
distinguishable  in  the  cells,  but  the  great  majority  of  them  occur 
as  small  bluish  indistinct  points  inside  the  cells.  To  demonstrate 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  129 

phagocytosis  well  boracic  carmine  followed  by  Gram's  stain  should 
be  used.* 

Fourth  stage.  Post  phagocytic  stage  or  stage  of  reinfection. — 
Preparations  from  the  exudate  34  hours  after  injection  still  show 
a  few  small  unphagocyted  bacteria,  but  whereas  these  organisms 
are  in  relatively  small  numbers,  the  number  of  well  stained,  plump 
and  normal  appearing  chains  is  considerably  increased.  That  is 
to  say  this  latter  form  of  the  streptococcus  has  increased  propor- 
tionately over  the  other.  New  streptococci  have  been  formed  by 
the  multiplication  of  the  normal  appearing  organisms  that  have 
resisted  engulfing.  The  animal  dies  shortly  after,  say  40  hours  after 
inoculation. 

A  few  scattered  streptococci  in  short  chains  and  normal  in 
appearance  are  found  in  the  blood,  as  is  usual  at  the  time  of  death 
in  rabbits  injected  with  serum. 

This  description  of  a  peritoneal  infection  characterized  by  a 
partial  phagocytosis  naturally  varies  considerably.  The  duration 
of  the  different  stages  may  vary  and  the  relative  proportion  of 
the  normal  and  abnormal  bacteria  before  phagacytosis  may  also 
change.f 

Sometimes  the  animal,  being  exhausted,  dies  at  the  beginning  of 
a  phagocytosis  that  concerns  only  a  small  proportion  of  the  bac- 
teria present  in  the  exudate.  But  one  observation  is  constant, 
namely,  a  peculiar  appearance  of  the  bacteria  at  the  moment  when 
phagocytosis  begins;  this  peculiarity  of  faint  staining  and  small- 
ness  which  is  found  in  the  majority  of  bacteria  that  are  being  taken 
up  by  phagocytes  is  shown  best  by  staining  with  methylene  blue. 

*  Staining  bacteria  with  gentian  violet  followed  by  Gram's  stain  and  decolori- 
zation  with  alcohol  and  clove  oil  is  not  always  satisfactory.  It  stains  bacteria 
rather  violently  and  does  not  bring  out  the  differences  between  individual  strep- 
tococci. To  bring  out  clearly  details  in  the  appearance  of  bacteria  a  more  delicate 
stain  like  Kiihne's  blue  is  necessary. 

The  normal  appearing  chains  already  mentioned  are  rarely  to  be  found  within 
cells.  They  remain  extracellular  and  are  somewhat  increased  in  number,  whereas 
the  number  of  altered  bacteria  has  greatly  decreased  outside  the  cells  owing  to 
phagocytosis. 

t  Partial  phagocytosis  can  appear  only  relatively  late.  For  example,  we  have 
seen  a  partial  phagocytosis  appear  after  nearly  48  hours.  But  even  in  this 
instance  the  exudate  was  full  of  leucocytes  at  12  hours  and  later  became  purulent. 
Such  cases  prove  the  extreme  persistence  of  the  negative  chemiotaxis  of  the 
micro-organism. 


130  STUDIES  IN   IMMUNITY. 

Another  point  which  constantly  recurs  is  the  more  or  less  distinctly 
superior  phagocytic  activity  of  the  mononuclears  over  the  mi- 
crophages  particularly  to  be  noted  in  beginning  phagocytosis. 
Another  regular  finding  is  a  relation  between  the  consistency  of  the 
exudate  and  the  occurrence  of  phagocytosis. 

In  exudates  containing  large  numbers  of  leucocytes  and  bac- 
teria there  are  often  to  be  found,  before  partial  phagocytosis  begins, 
localities  where  cells  are  collected  in  more  or  less  compact  masses; 
these  leucocytes  frequently  show  changes;  their  protoplasm  is  not 
distinctly  outlined  and  they  are  frequently  confluent;  these  clumps 
are  surrounded  by  a  slimy  layer  that  does  not  stain  well ;  this  layer 
is  apparently  mucilaginous  and  has  some  distinct  relation  to 
cellular  disintegration.  Within  this  layer  there  are  large  numbers 
of  streptococci  to  be  found  that  are  small  and  poorly  colored,  in 
other  words  that  show  distinct  evidence  of  abnormality,  having 
apparently  developed  there  and  been  retained  by  the  more  fluid 
nature  of  the  exudate  at  this  point. 

When  taken  from  the  body  the  exudate  coagulates,  the  leucocytes 
are  killed  and  the  fluid  therefore  becomes  distinctly  bactericidal. 

B.  Delayed  complete  phagocytosis. — In  order  for  a  complete 
phagocytosis  to  take  place  it  is  necessary  for  the  bacteria  to  be  in 
not  too  great  numbers  and  for  the  animal  not  to  be  too  exhausted. 
The  pre-phagocytic  period  is  similar  to  the  corresponding  period 
in  incomplete  phagocytosis,  but  is  not  so  long.  When  the  animal 
survives,  a  progressive  diminution  in  the  number  of  living  bac- 
teria within  the  phagocytes  is  to  be  noted  day  by  day;  the  cocci 
inside  the  phagocytes  are  separated. 

The  phagocytic  activity  in  complete  phagocytosis  is  very  great, 
not  only  on  the  part  of  the  mononuclears,  but  also  by  the  polynu- 
clears.  The  macrophages  do  not  content  themselves  with  taking 
up  streptococci,  but  also  take  up  more  or  less  degenerated  polynu- 
clears,  which  may  already  themselves  have  taken  up  bacteria.  This 
point  has  already  been  brought  out  by  Metchnikoff  in  studying  the 
phagocytosis  of  the  streptococcus  in  erysipelas. 

There  are  all  transitions  between  delayed  phagocytosis  and 
immediate  phagocytosis.  The  latter,  as  we  have  seen,  occurs  in 
rabbits  immunized  by  serum  and  prepared  by  bouillon  on  the  intra- 
peritoneal  injection  of  a  small  amount  of  streptococcus.  Without 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  131 

being  absolutely  instantaneous,  phagocytosis  in  these  cases  is  so 
rapid  as  to  prevent  any  extracellular  reproduction  of  the  strepto- 
coccus. In  addition,  cases  are  met  with  in  which  the  phagocytic 
crisis,  although  somewhat  delayed,  is  yet  so  rapid  that  no  exuberant 
development  of  the  bacterium  takes  place;  we  have  found  such 
phagocytosis  10  hours  after  inoculation.  Cases  like  this  show  a 
connection  between  the  two  forms  of  phagocytosis  and  it  seems 
logical  to  admit  that  if  the  leucocytes  in  prepared  rabbits,  with 
small  doses  of  culture,  can  rapidly  take  up  all  the  bacteria  inocu- 
lated, that  it  is  owing  to  a  mechanism  identical  with  that  which 
brings  about  delayed  phagocytosis. 

As  we  have  already  seen,  phagocytosis  may  be  brought  about  by 
injecting  into  the  peritoneal  cavity  of  a  rabbit  previously  prepared 
with  bouillon,  a  mixture  of  preventive  serum  and  young  strepto- 
coccus culture.  The  question  arises  whether  the  accomplishment 
of  this  delayed  phagocytosis  is  favored  or  accelerated  when  the  mix- 
ture of  bacteria  and  serum  injected  has  been  in  contact  for  several 
hours;  or  whether  the  phagocytic  phenomena  are  as  distinct  and 
as  rapid  when  the  two  factors  are  injected  separately  into  the  peri- 
toneal cavity  without  having  been  previously  mixed. 

Let  us  take  two  rabbits  of  the  same  weight  and  prepare  them 
by  injecting  6  c.c.  of  pepton  bouillon  into  the  peritoneal  cavity  of 
each.  On  the  following  day  rabbit  A  is  given  4  c.c.  of  preventive 
serum  intraperitoneally ;  at  this  time  of  course  the  peritoneum  con- 
tains many  leucocytes.  Rabbit  B  is  given  4  c.c.  of  normal  horse 
serum  at  the  same  time.  There  have  been  prepared  a  few  hours 
before  two  mixtures  composed  as  follows:  No.  1,  4  c.c.  of  preven- 
tive serum  and  0.5  of  a  cubic  centimeter  of  young  streptococcus 
culture;  No.  2,  4  c.c.  of  normal  horse  serum  and  0.5  of  a  cubic 
centimeter  of  the  same  streptococcus  culture.*  These  mixtures 
have  been  kept  at  room  temperature.  At  times  varying  from  one- 
half  or  three-quarters  of  an  hour  to  seven  or  eight  hours  after  the 
first  injection  of  rabbits  A  and  B,  these  two  mixtures  may  be  in- 
jected into  the  respective  peritoneal  cavities  as  follows:  rabbit  A; 
that  has  received  preventive  serum,  is  given  the  mixture  containing 
normal  serum.  Rabbit  B  that  has  received  the  injection  of  normal 
horse  serum,  subsequently  receives  the  mixture  containing  preven- 
*  We  have  varied  these  doses  in  different  experiments. 


132  STUDIES  IN   IMMUNITY. 

tive  serum.  Both  these  rabbits  have  received,  then,  in  the  same 
region  the  same  amounts  of  bacteria  and  of  serum;  they  differ,  in 
that  one  has  received  both  substances  separately  and  the  other 
received  them  at  the  same  time. 

Rabbit  B,  that  received  preventive  serum  plus  bacteria  subjected 
to  it,  not  only  survives  longer,  but  shows  the  most  complete  and 
accelerated  phagocytosis.  Neither  rabbit  recovers,  as  the  amount 
of  bacteria  given  was  purposely  too  large  for  the  dose  of  serum, 
but  both  live  longer  than  the  control.  In  correspondence  with 
the  greater  rapidity  of  phagocytosis  in  rabbit  B  the  total  develop- 
ment of  streptococci  has  remained  distinctly  more  restricted ;  before 
phagocytosis  occurs  the  peculiarities  already  noted  in  the  bacteria 
are  shown  more  clearly. 

This  experiment,  which  we  have  repeated  a  number  of  times, 
gives  the  same  results  uniformly.  The  same  results  are  also  ob- 
served in  experimenting  on  rabbits  that  have  not  been  previously 
prepared  by  means  of  bouillon. 

It  is  to  be  noted,  however,  that  the  differences  between  the  two 
rabbits  in  the  experiment  cited  are  only  relative  differences.  In 
both  animals  phagocytosis  is  delayed,  but  it  appears  more  readily  in 
the  one  than  in  the  other.  At  the  beginning  of  the  experiment,  dur- 
ing the  first  hours,  however,  the  phenomena  are  similar  and  the  bac- 
terial growth  goes  on  actively  in  both.  A  previous  contact  between 
the  bacterium  and  the  serum  in  vitro  may  favor  a  cure,  but  even  pro- 
longed contact  with  the  serum  apparently  causes  no  modification  in 
the  micro-organism  evident  after  its  injection  into  the  animal. 

IV.  INTRAVENOUS,  INTRAOCULAR  AND  SUBCUTANEOUS  INOCU- 
LATION OF  THE  STREPTOCOCCUS  IN  RABBITS  TREATED  BY 
SERUM. 

We  have  already  seen  that  a  dose  of  0.1  to  0.25  of  a  cubic  cen- 
timeter of  streptococcus  culture  injected  intravenously  in  animals 
vaccinated  by  serum  does  not  increase  to  any  great  extent  in  the 
body.  It  is  evident,  however,  that  the  injected  bacteria  live,  as  the 
animal  dies  in  2  to  4  days.  At  autopsy  few  bacteria  are  found  in 
the  blood,  but  more  are  present  in  the  liver,  spleen,  bone  marrow 
and  particularly  the  lung. 

If  a  small  number  of  bacteria  are  injected  in  the  first  place,  it  is 


STUDY  OF  ANTISTREPTOCOCCUS  SERUM.  133 

difficult  to  find  them  microscopically.  It  is  quite  probable  that 
these  organisms  have  been  taken  up  by  leucocytes  in  the  blood,  since 
the  serum  of  animals  that  have  received  preventive  serum  shows  no 
bactericidal  property  that  apparently  would  offer  resistance  to  the 
growth  of  the  micro-organism  in  the  circulating  blood.  We  know, 
to  be  sure,  that  when  small  amounts  of  streptococci  are  inoculated 
into  a  region  containing  many  leucocytes  (a  prepared  peritoneum) 
they  are  rapidly  taken  up  and  their  growth  inhibited.  Moreover, 
at  autopsy  of  such  rabbits  after  death  there  are  frequently  to  be 
found  in  the  internal  organs  (lungs  or  spleen)  leucocytes,  particularly 
mononuclears,  containing  numerous  streptococci. 

A  subcutaneous  inoculation  is  the  one  most  easily  tolerated  by 
animals  that  have  received  serum.  No  edema  occurs  at  the  point 
of  inoculation  (except  with  inoculations  in  the  ear),  which  renders 
the  study  of  this  condition  rather  difficult.  The  bacteria  do  not  pass 
into  the  blood  and  it  is  probable  that  the  cells  of  the  lymph  spaces 
in  the  glands  have  an  important  function  in  defending  the  body. 
We  have  many  things  still  to  consider  on  this  point.  Denys  and 
Leclef,  particularly  by  studying  the  effects  of  subcutaneous  inocu- 
lations in  the  ear,  have  established  the  fact  that  the  immunity  is 
due  to  phagocytosis.  Inoculation  into  the  aqueous  humor  is  dan- 
gerous. The  very  smallest  amount  of  streptococci  when  introduced 
here  increases  rapidly.  The  influx  of  leucocytes  is  slow,  not  occur- 
ring to  any  extent  under  24  hours,  and  is  not  sufficient  to  protect  the 
animal  for  any  length  of  time.  Later  on  well  stained  chains  of  strep- 
tococci surrounded  by  an  areola  are  found  free  amid  leucocytes  that 
contain  few  bacteria;  a  generalized  infection  finally  takes  place. 
The  serum  injected  into  such  a  rabbit,  however,  was  very  active; 
as  it  protected  another  rabbit  in  the  same  dose  against  the  sub- 
cutaneous inoculation  of  0.25  of  a  cubic  centimeter  of  a  young  and 
very  virulent  culture. 


VI.    ON  THE  AGGLUTINATION  AND   DISSOLUTION   OF 

RED  BLOOD  CELLS  BY  THE'  SERUM  OF  ANIMALS 

INJECTED  WITH   DEFIBRINATED  BLOOD  * 

BY  DR.   JULES   BORDET. 

In  an  article  published  in  1895f  we  called  attention  to  the 
following  facts: 

First: — The  serum  of  animals  which  have  been  vaccinated  against 
the  cholera  vibrio  causes  a  remarkable  phenomenon  when  mixed 
with  a  culture  of  vibrios  suspended  in  salt  solution  or  bouillon. 
A  very  small  dose  of  this  serum  will  cause  a  loss  of  motility  in  the 
bacteria  and  will  collect  them  in  masses  or  clumps.  If  the  serum 
has  been  freshly  obtained  and  added  in  a  sufficient  dose  to  the 
emulsion  its  action  on  the  bacteria  is  still  more  extensive.  The 
vibrios  that  have  been  clumped  are  soon  transformed  into  granules 
identical  with  those  observed  by  Pfeiffer  in  the  peritoneal  cavity  of 
immunized  guinea-pigs  on  injecting  a  culture,  and  the  same  as 
those  produced  by  Metchnikoff  in  vitro  by  mixing  an  emulsion 
of  vibrios,  preventive  serum,  and  peritoneal  exudate  containing 
leucocytes.  This  granular  transformation  is  the  visible  indication 
of  an  extensive  destruction  of  the  bacteria. 

Second :  —  Serum  kept  for  some  time  or  heated  to  55  degrees  loses 
its  power  to  produce  a  granular  transformation  in  vibrios  but  still 
clumps  them.  This  clumping  always  occurs  in  the  presence  of 
preventive  serum  and  may,  therefore,  be  very  marked  with  a  serum 
deprived  of  its  bactericidal  activity. 

We  may  note  that  Fraenkel  and  Sobernheim  (Hygienische  Rund- 
schau, January,  1894)  had  already  noted  that  cholera  serum  heated 
to  55  degrees  or  even  as  high  as  GO  degrees  or  70  degrees  C.  loses  its 
bactericidal  power  but  retains  its  preventive  power. 

*  Sur  1'agglutination  et  dissolution  des  globules  rouges  par  le  serum  d'animaux 
injected  de  sang  defibrine*.     Annales  de  1'Institut  Pasteur,  1898,  XII,  688. 
t  See  p.  8. 

134 


AGGLUTINATION  AND   DISSOLUTION.  135 

Third :  —  If  we  add  the  fresh  serum  of  a  normal  animal  to  cholera 
serum  previously  heated  to  55  degrees,  and  which  consequently 
no  longer  effects  a  granular  transformation  but  still  clumps  the 
vibrios,  we  find  that  the  bactericidal  power  is  restored  to  the  pre- 
ventive serum,  that  is,  it  again  produces  granules ;  and  yet  heated 
preventive  serum  alone  is  an  excellent  culture  medium,  and  normal 
serum  alone  has  only  slight  bactericidal  power.  In  other  words, 
the  two  constituents  of  the  mixture,  separately,  are  slightly,  if  at 
all,  bactericidal;  when  united,  however,  they  act  energetically  on  the 
vibrio.  Normal  serum  restores  to  the  preventive  serum  that  sub- 
stance which  heat  has  destroyed,  but  it  is  incapable  of  restoring 
this  property  when  itself  heated  to  55  degrees.  It  is  rather  striking 
to  note  how  very  small  an  amount  of  preventive  serum,  whether 
fresh  or  heated  to  55  degrees,  will  endow  normal  serum  with  strong 
bactericidal  activity.  From  these  facts  we  concluded  that  the 
intense  destructive  power  against  bacteria  present  in  immune  serum 
is  due  to  the  action  of  two  distinct  substances,  one  of  which  is 
characteristic  of  immunized  animals,  is  endowed  with  specificity, 
is  capable  of  acting  in  a  very  small  dose,  and  resists  heat;  and  the 
other  of  which  is  present  in  normal  as  well  as  immunized  animals, 
is  destroyed  by  heating  to  55  degrees,  is  not  in  itself  specific,  and 
has  only  a  slight  activity  when  not  associated  with  the  first  substance. 
Without  indulging  in  hypotheses  as  to  the  intimate  mechanism  of 
the  action  of  the  two  substances,  we  offered  as  a  probable  explana- 
tion that  the  specific  substance  by  immobilizing  and  clumping 
bacteria  renders  them  more  susceptible  to  the  bactericidal  sub- 
stance (alexin)  present  in  the  serum  of  normal  as  well  as  of  immun- 
ized animals. 

It  may  easily  be  understood  then  why  the  injection  in  normal 
animals,  of  either  fresh  or  heated  cholera  serum,  gives  rise  to  a 
specific  bactericidal  power  in  their  serum ;  *  the  specific  substance 
unites  in  the  injected  animal  with  the  alexin  already  present.  The 
serum  obtained  from  an  animal  after  such  an  injection  contains, 
therefore,  the  two  substances,  the  presence  of  both  of  which  is  neces- 
sary to  affect  the  vibrio  seriously  as  indicated  by  granular  metamor- 

*  We  noted  in  1895  that  if  a  normal  guinea-pig  is  injected  with  preventive 
serum  active  against  the  vibrio  Metchnikovi,  the  serum  of  this  animal  becomes 
bactericidal  for  the  vibrio  Metchnikovi,  but  not  for  the  cholera  vibrio. 


136  STUDIES  IN   IMMUNITY. 

phosis.  This  theory  of  "two  substances"  as  explanatory  of  the 
origin  of  the  bactericidal  power  in  the  serum  of  passively  immunized 
animals  was  accepted  in  the  following  year  by  Gruber  and  Durham ; 
we  shall  return  presently  to  this  subject  and  particularly  to  certain 
objections  that  Pfeiffer  has  made  to  our  theory. 

Other  facts  were  soon  added  to  those  just  mentioned.  In  the 
early  part  of  1896  both  Gruber  and  we  ourselves  recognized  that 
the  property  of  immobilizing  and  clumping  bacteria  was  not  found 
exclusively  in  the  serum  of  immunized  animals.  We  noted,  for 
example,  that  normal  horse  serum  clumps  the  cholera  vibrio,  B. 
coli,  B.  typhosus  and  B.  tetani  very  distinctly.  The  serum  of 
other  animals  also  agglutinates  them,  but  usually  to  a  less  extent. 
This  property  of  agglutination  which  is  so  marked  in  immunized 
animals  occurs,  as  it  were,  in  a  primitive  condition  in  the  serum  of 
normal  animals.  We  also  called  attention  in  1895  and  subsequently* 
to  the  fact  that,  as  a  general  rule,  the  serum  of  one  animal  will  clump 
the  red  blood  cells  of  an  animal  of  a  different  species.  This  power 
also  is  frequently  present  to  a  very  marked  extent;  thus  it  is  found 
that  the  serum  of  the  hen  clumps  rat  corpuscles  and  especially 
rabbit  corpuscles  with  surprising  energy.  Thanks  to  the  researches 
of  Prof.  Buchner  we  have  known  for  some  time  that  a  given  serum 
is  frequently  able  to  destroy  the  red  blood  cells  of  an  animal  of 
another  species  by  diffusing  their  hemoglobin  and  so  making  them 
transparent;  a. good  example  of  this  phenomenon  is  the  action  of 
rabbit  serum  on  guinea-pig  red  blood  corpuscles.  Buchner  also 
showed  that  a  temperature  of  55  degrees  will  destroy  this  destructive 
power  for  red  blood  cells  in  serum  as  well  as  the  analogous  power 
for  bacteria. 

It  is  easy  to  determine  that  these  two  phenomena  of  clumping 
and  of  destruction  of  corpuscles  by  serum  from  a  different  animal 
species  are  due  to  two  separate  substances.  The  destructive  sub- 
stance which  causes  corpuscles  to  lose  their  hemoglobin  is  destroyed 
at  55  degrees,  as  Buchner  showed,  but  the  clumping  substance  re- 
sists heating  to  this  temperature.  In  experiments  of  this  kind  I 
have  usually  heated  the  sera  to  55  degrees  for  half  an  hour.  For 
example,  we  find  that  fresh  hen  serum  agglutinates  and  then  destroys 
rabbit  corpuscles ;  when  heated  to  55  degrees  it  still  clumps  them  as 

*  See  page  92. 


AGGLUTINATION   AND  DISSOLUTION.  137 

well  as  before,  but  does  not  destroy  them,  as  is  shown  by  the  fact 
that  they  retain  their  color  and  normal  appearance. 

It  is  evident  that  there  is  a  striking  parallel  between  those  changes 
shown  by  vibrios  subjected  to  cholera  serum  and  those  in  red  blood 
cells  affected  by  serum  from  an  alien  species.  We  have  noted  that 
the  clumping  effect,  which  is  more  or  less  evident  in  both  instances, 
is  due  to  substances  which  resist  heating  to  55  degrees  or  even 
more;  it  has  also  been  noted  that  the  destructive  properties  neces- 
sitate the  presence  of  a  more  susceptible  substance,  which  is  de- 
stroyed by  heating  to  55  degrees.  In  a  general  way  certain  analogies 
are  present  in  the  sera  of  normal  animals,  as  a  weak  clumping  power 
both  for  bacteria  and  red  blood  cells  is  frequently  found  in  them; 
normal  sera,  moreover,  have  usually  some  altering  or  destructive 
effect  both  for  alien  red  blood  cells  and  susceptible  micro-organ- 
isms. As  has  already  been  noted,  the  cholera  vibrio  if  attenuated 
and  only  slightly  resistant  may  show  at  least  partial  transformation 
with  a  normal  serum. 

If  a  normal  animal  is  vaccinated  with  the  cholera  vibrio,  the 
original  clumping  and  destructive  properties  of  its  serum  are  con- 
siderably increased.*  On  account  of  the  parallelism  that  we  have 
just  indicated  and  on  account  of  analogies  in  the  action  of  sera  on 
cells  and  bacteria,  a  question  immediately  arises.  Would  it  be 
possible  by  injecting  a  normal  animal  with  the  defibrinated  blood 
of  an  animal  of  a  different  species  to  increase  the  clumping  and  de- 
structive property  of  its  serum  for  the  corpuscles  injected?  Experi- 
ment gives  a  positive  answer  to  this  question.  Guinea-pigs  were 
injected  intraperitoneally  five  or  six  times  with  10  c.c.  of  defibri- 
nated rabbit  blood. f  The  animals  stand  this  treatment  very  well. 
After  a  time  their  blood  is  withdrawn  and  the  serum  is  found  to  have 
the  following  characteristics: 

First:  —  When  added  to  defibrinated  rabbit  blood  it  clumps  the 
red  corpuscles  energetically.  For  example,  one  part  of  serum  will 

*  We  use  the  cholera  vibrio  as  an  example,  although  it  is  well  known  that 
vaccination  with  other  bacteria  will  also  cause  the  appearance  of  agglutinating 
power.  The  cholera  vibrio,  however,  on  account  of  its  susceptibility  is  the  best 
organism  to  demonstrate  the  bactericidal  substance. 

t  These  doses,  first  used  by  Dr.  Bordet  to  produce  a  hemolytic  serum,  have  since 
been  found  in  his  hands,  as  well  as  those  of  other  investigators,  to  be  unnecessarily 
large.  Quite  as  good  results  can  be  obtained  by  injecting  much  smaller  amounts, 
for  example,  1  c.c.  (Ed.) 


138  STUDIES   IN   IMMUNITY. 

clump  the  red  blood  cells  in  fifteen  parts  of  defibrinated  rabbit 
blood. 

Second :  —  The  corpuscles  that  have  been  clumped  by  this  serum 
subsequently  undergo  rapid  and  complete  destruction;  for  example, 
in  a  mixture  of  one  part  of  defibrinated  rabbit  blood  and  two  or 
three  parts  of  active  serum  the  mixture  becomes  red  and  perfectly 
transparent  in  2  or  3  minutes.  Microscopically,  nothing  but 
the  stromata  of  the  corpuscles  are  found  in  the  fluid;  they  appear 
more  or  less  distorted,  very  transparent,  without  their  usual  sheen, 
and  rather  difficult  to  discern. 

Third :  —  If  this  active  guinea-pig  serum  is  heated  to  55  degrees  for 
half  an  hour  it  loses  the  property  of  destroying  rabbit  corpuscles, 
but  still  agglutinates  them. 

Fourth :  —  if  a  certain  quantity  of  fresh  normal  guinea-pig  serum 
is  added  to  a  mixture  of  defibrinated  rabbit  blood  and  specific  serum 
heated  to  55  degrees,  the  phenomena  of  destruction  reappear  in 
their  entirety.  The  mixture  becomes  limpid  and  red  in  a  few 
minutes.  It  is  rather  surprising  to  find,  moreover,  that  the  experi- 
ment succeeds  perfectly  if  fresh  serum  from  the  very  rabbit  whose 
corpuscles  are  affected  is  added  to  the  mixture  of  corpuscles  and 
heated  specific  serum.  That  is  to  say,  the  corpuscles  of  this  rabbit 
have  become  susceptible  to  their  own  alexin  under  the  influence 
of  the  foreign  clumping  substance  from  a  guinea-pig  treated  with 
defibrinated  rabbit  blood. 

Fifth :  —  Although  it  is  true  that  active  guinea-pig  serum  loses  its 
destructive  property  by  heating  to  55  degrees  it  is  not  quite  exact 
to  say  that  defibrinated  rabbit  blood  when  mixed  with  such  a 
serum  remains  wholly  intact.  There  is  sufficient  destruction  of  red 
blood  cells  to  give  the  fluid  a  more  or  less  reddish  color,  although 
the  destruction  is  only  partial  and  very  slow.  This  destruction  is 
due  to  the  fact  that  the  defibrinated  blood  contains,  not  only 
corpuscles,  but  also  serums  containing  a  certain  amount  of  alexin, 
and,  as  we  have  just  seen,  normal  rabbit  alexin  will  act  on  rabbit 
corpuscles  when  the  latter  have  been  affected  by  the  clumping  sub- 
stance of  active  serum.  The  amount  of  alexin  in  the  defibrinated 
rabbit  blood,  however,  is  not  sufficient  to  destroy  a  large  number 
of  corpuscles,  which  explains  why  the  destruction  of  red  blood  cells 
is  very  slow  and  only  partial  in  such  a  mixture. 


AGGLUTINATION  AND  DISSOLUTION.  139 

Sixth :  — The  phenomena  mentioned  do  not  occur  if  normal  guinea- 
pig  serum  is  used  instead  of  the  serum  from  a  guinea-pig  that  has 
been  treated  with  frequent  injections  of  defibrinated  rabbit  blood. 
Normal  guinea-pig  serum  has  only  a  very  slight  clumping  effect  on 
rabbit  corpuscles  and  its  destructive  power  against  them  is  practi- 
cally nil. 

Seventh :  —  The  specific  serum  of  a  treated  guinea-pig  has  no  effect 
on  the  defibrinated  blood  of  a  normal  guinea-pig.  It  has,  moreover, 
no  effect  on  the  red  blood  cells  of  the  pigeon.  It  agglutinates  ener- 
getically rat  and  mouse  corpuscles,  but  no  more  so  than  normal 
guinea-pig  serum.  This  guinea-pig  serum,  which  affects  rabbit 
blood,  has  slightly  more  destructive  properties  for  rat  and  mouse 
corpuscles  than  does  normal  guinea-pig  serum;  but  the  destruction 
of  corpuscles  in  a  mixture  of  this  active  serum  and  rat  or  mouse 
blood  is  very  much  less  complete  and  rapid  than  in  a  mixture 
of  the  serum  with  rabbit  corpuscles.  We  intend  trying  the  effect 
of  this  serum  on  the  corpuscles  of  a  great  number  of  species  in 
order  to  determine  just  how  far  the  phenomenon  is  specific;  the 
specificity,  however,  from  the  data  'that  we  have  already  given 
would  seem  to  be  very  distinct,  if  not  absolute. 

Eighth:  —  If  a  small  amount  (2  c.c.  for  example)  of  defibrinated 
rabbit  blood  is  injected  into  the  peritoneal  cavity  of  a  treated  guinea- 
pig  (that  is  a  guinea-pig  that  has  received  several  injections  of 
rabbit  blood)  the  corpuscles  are  rapidly  destroyed.  The  fluid  with- 
drawn from  the  peritoneal  cavity  ten  minutes  later  is  red  and  limpid. 
^ he  corpuscles  remain  intact  much  longer  if  injected  subcutaneously. 
If  such  an  injection  is  made  into  the  peritoneal  cavity  of  a  normal 
guinea-pig  the  corpuscles  remain  unchanged  and  are  finally  taken 
up  by  the  macrophages. 

Ninth:  —  If  rabbit  blood  plus  a  small  amount  of  active  serum 
previously  heated  to  55  degrees,  is  injected  into  the  peritoneal 
cavity  of  a  normal  guinea-pig  a  similar  destruction  of  corpuscles 
occurs. 

Tenth: — As  might  be  expected  an  active  serum  with  so  marked  an 
effect  on  rabbit  corpuscles  is  toxic  for  this  animal.  Two  cubic  centi- 
meters injected  into  the  ear  vein  is  fatal.  We  shall  later  return 
to  a  discussion  of  the  symptoms  and  lesions  which  such  injections 
cause. 


140  STUDIES  IN   IMMUNITY. 

It  must  be  quite  evident  to  the  reader  how  close  an  analogy  there 
is  between  the  action  of  cholera  serum  and  of  this  anticorpuscular 
serum,  the  properties  of  which  we  have  merely  outlined.  In  the 
preceding  pages  the  description  so  much  resembles  the  description 
of  a  specific  cholera  serum  that  it  would  hold  for  the  latter  if  the 
words  "defibrinated  blood"  were  replaced  by  the  words  "culture 
of  vibrios"  and  the  expression  "destruction  of  rabbit  blood  cells" 
by  the  expression  "granular  transformation  of  the  vibrio."  The 
analogy  is  still  more  striking  when  we  consider  that  the  alexin  that 
affects  rabbit  blood  cells  is  probably  identical  with  that  which 
causes  a  granular  transformation  of  the  vibrios.  At  least  the 
intense  destructive  power  which  is  evident  in  both  instances  is 
destroyed  at  55  degrees.  In  both  instances  this  destructive  property 
would  seem  to  be  widely  distributed,  not  only  in  the  serum  but  in 
the  peritoneal  exudate,  and  it  would  seem  to  be  absent  from  sub- 
cutaneous edema  fluid  obtained  by  venous  compression.  If  mix- 
tures of  defibrinated  guinea-pig  blood  and  rabbit  serum  are  made 
on  the  one  hand  and  defibrinated  blood  and  edema  fluid  from  the 
same  rabbit  on  the  other  hand,  it  will  be  found  that  there  is  destruc- 
tion of  the  guinea-pig  corpuscles  in  the  serum  tube,  but  none  in  the 
edema  tube.  As  we  already  know  edema  fluid  also  fails  to  produce 
a  metamorphosis  of  vibrios  treated  with  heated  cholera  serum. 

What  conclusions  may  be  drawn  from  these  analogies?  We 
may  conclude  that  the  properties  with  which  cholera  serum  is 
endowed  have  not  been  manufactured  by  the  animal  body  for  the 
simple  purpose  of  combating  an  infection,  if  we  may  so  express 
it,  but  are  due  simply  to  the  starting  up  of  certain  preexistent 
functions  that  may  be  directed  according  to  chance  conditions  either 
against  such  harmful  substances  as  vibrios  or  else  against  such 
wholly  innocuous  elements  as  red  blood  cells.  As  we  have  already 
shown,  on  injecting  animals  with  harmless  substances  such  as  red 
blood  cells  we  obtain  a  serum  which  affects  these  cells  just  as  a 
cholera  serum  affects  the  cholera  vibrio.  In  the  case  of  the  cholera 
vibrio  these  properties  do  not  arise  spontaneously,  expressly  to 
defend  the  animal  against  this  organism,  any  more  than  does  phago- 
cytosis, the  very  keystone  of  immunity,  owe  its  existence  to  a  need 
of  combating  bacterial  infections.  One  of  the  most  important 
conclusions  to  be  drawn  from  Metchnikoff's  work  is  that  immunity 


AGGLUTINATION  AND  DISSOLUTION.  141 

is  simply  an  instance  of  intracellular  digestion  and  entirely  a 
chance  and  efficient  application  to  animal  defense  of  a  primitive 
function  which  would  exist  even  if  there  were  no  pathogenic  organ- 
isms in  existence.  Since  this  form  of  intracellular  digestion  is  so 
admirably  fitted  to  aid  in  the  survival  of  the  individual  it  has  been 
made  use  of  for  this  purpose. 


VII.    THE  MECHANISM  OF  AGGLUTINATION  * 

BY  DR.   JULES   BORDET. 

The  expression  "phenomenon  of  agglutination"  is  usually  em- 
ployed to  indicate  that  bacteria,  in  a  homogeneous  suspension  in  a 
fluid  like  bouillon  or  isotonic  salt  solution  collect  in  clumps  and 
fall  to  the  bottom  of  the  tube  when  acted  on  by  a  specific  serum. 
We  demonstrated  the  first  instance  of  this  phenomenon  in  1895 
by  showing  that  cholera  vibrios  suspended  in  salt  solution  lose 
their  motility  when  subjected  to  the  action  of  a  small  dose  of  fresh 
or  heated  anticholera  serum  and  that  they  then  rapidly  collect  in 
small  masses  that  float  in  the  fluid. f 

This  agglutination  must  be  considered  from  several  different  stand- 
points. In  the  first  place  it  must  be  studied  as  an  entity  without 
any  reference  to  its  physiological  significance.  When  looked  at 
from  this  viewpoint  agglutination  has  evident  relation  both  with 
physics  and  with  chemistry.  If  we  attempt  to  define  the  importance 
of  this  phenomenon  in  immunity,  when  we  wish,  for  instance,  to 
know  whether  it  is  functional  in  defense  of  the  animal  body  or  what 
cells  secrete  the  substances  that  cause  it  and  liberate  them  in  the 
serum  and  so  endow  it  with  its  particular  activity,  we  must  con- 
sider agglutination  from  the  physiological  standpoint. 

In  the  present  study  we  shall  consider  the  mechanism  of  agglu- 
tination and  shall  begin  by  reviewing  the  principal  theories  that  have 
been  offered  to  explain  the  phenomenon.  We  may  note  at  once 
that,  to  be  satisfactory,  a  theory  on  a  subject  like  this  should  have 
a  general  bearing,  and  should  not  deal  simply  with  the  agglutination 
of  bacteria.  Bacteria  are  not  the  only  cells  clumped  by  serum; 
the  agglutination  of  red  blood  cells  by  the  serum  of  an  animal  of 
another  species,  must  also  be  taken  into  consideration.  We  have 

*  Le  mdchanisme  de  1'agglutination.  Annales  de  1'Institut  Pasteur,  1899, 
XIII,  225. 

t  See  article,  p.  8. 

142 


THE   MECHANISM   OF  AGGLUTINATION.  143 

already  shown  that  a  specific  serum  with  very  marked  agglutinating 
properties  for  red  blood  cells  may  be  obtained  by  injecting  an 
animal  of  another  species  with  a  given  blood.*  Any  acceptable 
explanation  then  should  be  applicable,  not  only  to  the  agglutina- 
tion of  bacteria  but  also  to  the  agglutination  of  blood  corpuscles 
and,  as  we  shall  see  further  on,  to  the  agglutination  of  particles  of 
casein  suspended  in  milk. 

With  this  remark  we  may  consider  the  different  hypotheses  that 
have  been  proposed. 

1.  Gruber's   hypothesis.  —  Gruber    thinks   that   the   agglutinin 
changes  the  bacterial  substance  essentially.    According  to  his  in- 
terpretation it  renders  the  membrane  of  the  micro-organisms  more 
viscous  and  this  viscous  condition  of  the  superficial  part  of  the 
bacteria  causes  them  to  stick  together  and  explains  their  collection 
in  definite  clumps. 

This  conception  explains  well  enough  why  bacteria  that  have 
once  been  united  remain  together,  but  it  does  not  in  any  way  explain 
how  the  organisms  approach  each  other  to  form  the  clumps.  In 
explaining  the  fact  it  emphasizes  exclusively  the  structure  of  the  cells 
affected  by  the  agglutinin,  without  admitting  that  the  phenomenon 
may  be  explained  even  in  part  by  simple  physical  laws.  Since  this 
hypothesis  depends  entirely  on  the  supposed  existence  of  a  change 
in  a  cell  membrane,  namely,  a  swelling  accompanied  by  the  pro- 
duction of  an  adhesive  substance,  it  in  no  way  explains  the  agglu- 
tination of  inorganic  chemical  particles.  It  rules  out  any  relation 
between  the  agglutination  of  bacteria  and  the  possible  clumping 
of  chemical  precipitates  in  a  fluid. 

2.  Bordet's  hypothesis.  —  The  conception   which  we   gathered 
from  a  study  of  this  phenomenon  in  1896  is  essentially  different. 
It  appeared  to  us  that  in  the  case  of  the  agglutination  of  the  cholera 
vibrio  by  its  specific  serum  we  have  to  deal  with  a  phenomenon 
in  which  the  bacteria  play  only  a  passive  role  and  in  which  their 
vitality  is  not  concerned.     It  is  evident  that  motility  is  not  neces- 
sary, since  agglutination  occurs  not  only  in  bacteria  that  have  lost 
their  motility,  but  also  in  red  blood  cells  which  are  inert.     The 
passive  role  of  bacteria  is  still  further  evident  when  the  agglutination 
of  dead  micro-organisms  is  considered.     Gruber's  hypothesis,  more- 

*  See  article  p.  134. 


144  STUDIES   IN   IMMUNITY. 

over,  arouses  certain  other  objections,  to  which  we  shall  later  refer, 
and  it  appeared  to  us  that  agglutination  "is  due  to  some  phenom- 
enon of  molecular  physics.  The  slightest  effects  may  cause  chemi- 
cal precipitates  which  have  remained  uniformly  suspended  in  a 
fluid  to  fall  to  the  bottom  of  the  tube.  It  is  probable  that  serum 
acts  on  bacteria  by  changing  the  relations  of  molecular  attraction 
between  the  bacteria  and  the  surrounding  fluid." 

It  is  evident  that  this  interpretation  does  not  explain  the  inti- 
mate nature  of  the  phenomenon  any  more  than  does  Gruber's; 
it  simply  compares  particles  of  such  different  natures  as  bacteria, 
red  blood  cells  and  chemical  precipitates,  each  of  which  when  sus- 
pended in  a  fluid  may  be  brought  together  in  masses  by  certain 
influences.  Contrary  to  Gruber's  conceptions,  this  explanation 
implies  the  existence  of  analogies  between  the  various  forms  of 
agglutination  whatever  may  be  the  substances  agglutinated ;  it  pre- 
supposes the  predominant  intervention  of  physical  laws  in  the 
phenomenon. 

We  must  be  quite  clear  on  this  point.  Does  this  hypothesis 
mean  that  bacteria  when  affected  by  the  agglutinin  act  simply  as 
inert  particles  in  all  phases  of  the  phenomenon?  Certainly  not. 
Agglutinins  are  specific;  and  there  is,  moreover,  not  the  slightest 
doubt  that  they  act  directly  on  the  bacteria  since  these  cells  rapidly 
lose  their  motility  in  the  very  first  stages  of  the  phenomenon.  In 
the  first  phase  of  the  phenomenon  the  action  of  the  agglutinin 
evidently  takes  into  account  the  particular  biological  nature  of  the 
element  that  it  affects :  this  is  evident  since  it  affects  certain  organ- 
isms and  not  others.  But  the  subsequent  changes  which  bring 
the  affected  organisms  together  may  be  brought  about  by  the  slight- 
est modifications  in  the  active  substance  provided  they  are  sufficient 
to  change  the  relation  of  molecular  adhesion  between  bacteria  and 
fluid.  From  that  point  on,  according  to  the  hypothesis,  the 
biological  nature  of  the  substances  affected  would  no  longer  count. 
The  bacteria  are  thenceforth  agglutinated  according  to  physical 
laws  which  are  applicable  also  to  certain  inorganic  particles,  and  it 
is  unnecessary  to  suppose  the  presence  of  an  adhesive  substance  or 
of  sticky  or  viscous  membranes  to  explain  the  clumping  and  the 
adhesion  of  the  micro-organisms.  Gruber's  hypothesis  excludes 
physical  laws;  our  hypothesis  would  attribute  considerable  impor- 


THE  MECHANISM  OF  AGGLUTINATION.  145 

tance  to  them,  particularly  during  the  phase  of  the  phenomenon 
in  which  the  bacteria  which  have  been  affected  by  the  agglutinin 
are  still  scattered  and  are  beginning  to  collect  to  form  the  typical 
agglutination. 

These  two  hypotheses,  which  were  formulated  in  the  beginning 
of  studies  on  agglutination,  could  not  rest  on  a  very  firm  basis  from 
lack  of  sufficient  data.  An  experimental  fact  of  importance  was 
furnished  by  Kraus.*  Kraus  showed  that  if  the  serum  of  animals 
vaccinated  against  the  cholera  vibrio  is  mixed  with  a  limpid  filtered 
culture  of  this  organism  a  precipitate  is  formed  in  the  fluid.  This 
reaction  is  specific  and  does  not  occur  when  any  other  serum  is 
used  in  place  of  cholera  serum.  When  this  precipitate  has  been 
formed  it  soon  collects  in  small  clumps  that  recall  in  appearance 
masses  of  agglutinated  bacteria.  Kraus  also  showed  that  the  same 
result  could  be  obtained  with  other  bacteria  and  their  correspond- 
ing antisera. 

These  experiments  would  seem  to  corroborate,  a  priori,  the  second 
of  the  two  hypotheses  that  we  have  mentioned.  It  would  seem, 
moreover,  to  invalidate  Gruber's  hypothesis,  which  recognizes  as 
the  sole  cause  of  agglutination  a  structural  modification  of  the 
affected  cells.  These  experiments  indeed  show  that  a  flaky  pre- 
cipitation resembling  true  agglutination  can  be  formed  by  mixing 
with  serum  a  fluid  containing  no  definite  bacteria  but  simply  the 
materials  of  bacterial  disintegration.  This  fact  of  Kraus'  would 
seem,  then,  to  rule  out  Gruber's  theory  entirely. 

3.  Nicolle's  hypothesis^ —  Nicolle  has  a  somewhat  different 
idea.  He  has  confirmed  Kraus'  results  and  agrees  that  the  agglu- 
tinin precipitates  the  agglutinable  (or  agglutinated)  substance 
of  bacteria.^  He  thinks,  moreover,  that  this  agglutinable  sub- 
stance, which  in  old  cultures  may  become  diffused  into  the  surround- 
ing fluid,  is  present  in  large  amounts  in  the  membrane  of  the  outer 

*  Kraus,  K.  K.  Gesellschaft  der  Aertze  in  Wien,  April  30,  1897,  and  Wiener 
klinische  Wochenschrift,  August  12,  1897,  No.  32. 

t  Annales  de  1'Institut  Pasteur,  March,  1898. 

J  This  is  more  than  a  simple  adaptation  of  Kraus'  experiment;  the  expression 
indicates,  perhaps  rather  hastily,  that  we  must  consider  the  substance  precipitated 
as  the  one  which  is  of  importance  in  the  agglutination  of  bacteria,  the  substance 
which,  in  other  words,  represents  in  bacteria  that  part  which  is  susceptible  to  the 
agglutinin. 


146  STUDIES  IN   IMMUNITY. 

surface  of  young  and  healthy  bacteria.  This  superficial  layer 
includes  the  substance  that  is  susceptible  to  attack  and  precipi- 
tation by  the  agglutinin;  when  the  agglutinin  acts,  this  external 
layer  of  the  bacteria  "swells  up,  becomes  apparent  and  sticks  to 
the  external  layer  of  adjacent  bacteria.  Our  opinion  as  to  the 
intimate  nature  of  the  phenomenon  of  agglutination  is  quite  similar, 
then,  to  that  offered  by  Gruber  and  later  defended  by  Roger.  We 
believe  that  agglutination  consists  in  the  coagulation  and  coales- 
cence of  the  external  layers  of  the  agglutinable  bacteria  by  the 
agglutinating  serum. "  * 

As  may  be  seen,  Nicolle  attaches  or  adds  on  Kraus'  experiment 
to  Gruber's  theory,  but  it  is  precisely  this  addition,  on  which  the 
whole  value  of  the  idea  depends,  that  appears  to  us  incomprehen- 
sible and  the  weak  point  in  the  reasoning.  Why  should  a  precipi- 
tation of  the  agglutinable  substance  within  the  external  layer  of 
the  bacterium,  which  we  will  not  deny  off  hand,  lead  to  a  swelling 
and  viscosity  which  may  bring  about  the  coalescence  and  junction 
of  the  external  layers  of  neighboring  organisms? 

However  this  may  be,  this  interpretation,  as  well  as  Gruber's, 
takes  no  account  of  the  intervention  of  the  physical  laws  of  molec- 
ular adhesion  in  explaining  the  fact.  Nor  does  it  presuppose 
any  relation  between  the  collection  of  certain  chemical  precipi- 
tates and  bacterial  agglutination,  since  its  explanation  of  the  phe- 
nomenon depends  essentially  on  the  presence  of  a  membrane  and 
external  layer  or  of  a  ciliated  covering  susceptible  to  swelling  and 
stickiness. 

Nicolle  describes  a  rather  curious  experiment  in  his  article.  He 
found  that  the  precipitate  caused  by  the  interaction  of  an  active 
serum  with  a  culture  filtrate  has  the  property  of  carrying  down  with 
it,  in  its  clumping,  inert  particles  like  talcum  powder  in  the  form  of 
definite  masses.  Although  this  experiment  is  interesting  we  do 
not  think  it  is  of  any  direct  importance  in  explaining  the  phenome- 
non of  agglutination,  its  resemblance  to  which  is  only  apparent. 
These  particles  of  talcum  that  collect  into  masses  arc  drawn  together 
mechanically  and  collected  by  a  precipitate  which  is  forming.  To 
admit  that  this  non-specific  phenomenon,  which  resembles  super- 
ficially true  agglutination,  is  of  importance,  would  be  to  admit 
*  Loc.  cit.,  page  191. 


THE  MECHANISM  OF  AGGLUTINATION.  147 

that  agglutination  proper  is  also  due  to  the  formation  of  a  pre- 
cipitate outside  of  the  bacteria  which  retracts  and  becomes  agglu- 
tinated and  thus  presses  together  the  bacteria  and  forces  them  to 
unite  and  become  adherent.  As  a  matter  of  fact  this  idea  was 
already  offered  by  Paltauf  *  before  Nicolle's  work.  Dineur  in  a 
recent  article  has  discussed  this  theory  and  offered  objections  to 
it.  f  We  shall  consider  this  hypothesis  in  its  proper  place. 

4.  Paltauf 's  hypothesis.  —  According  to  this  author  the  agglu- 
tination of  bacteria  is   due   to  their   being  mechanically  drawn 
together  in  the  interstices  of  a  coagulum  formed  outside  of  the  bac- 
teria in  the  surrounding  fluid,  as  a  result  of  the  reaction  between 
the  agglutinin  and  the  agglutinable  substances  from  the  bacteria. 

5.  Dineur' s  hypothesis.  —  According  to  Dineur  the  clumping  is 
due  to  the  formation  of  an  adhesive  substance  which  keeps  the 
bacteria  together.    This  adhesive  substance  is  formed  particularly 
on  the  cilia.     As  may  be  seen,  Dineur  attributes  essential  import- 
ance in  agglutination  to  the  presence  of  cilia.     Agglutination  would 
be  caused,  then,  by  an  adhesion  and  interlacing  of  these  cilia. 

*** 

We  have  thus  reviewed  in  the  preceding  pages  the  various  in- 
terpretations of  agglutination  that  have  been  proposed,  with  em- 
phasis on  their  exact  significance.  We  have,  however,  only  touched 
on  the  experimental  facts  that  corroborate  or  eliminate  them. 
There  are  already  enough  of  these  facts  to  allow  of  a  discussion 
founded  on  adequate  data,  and  it  is  this  consideration  that  we  pro- 
pose to  take  up. 

Among  the  interpretations  that  have  been  offered  there  are  some 
which  evidently  are  so  little  in  relation  to  fact  that  they  may  be 
dismissed  pree'mptorially.  For  example,  Dineur's  hypothesis  which 
attributes  a  maximum  importance  to  the  existence  of  cilia.  Such 
an  opinion  is  evidently  unsatisfactory,  as  agglutination  may  occur 
with  bacteria  that  have  no  cilia  or  even  with  such  elements  as  red 
blood  ceils,  or  particles  of  casein  which  obviously  do  not  possess 
these  appendages.  Dineur,  to  be  sure,  also  emphasizes  the  produc- 
tion of  an  adhesive  substance  which  collects  the  bacteria  subjected 

*  Wiener  klinische  Wochenschrift,  1897. 

t  Dineur,  Recherches  sur  le  mecanismede  1'agglutination  du  bacille  typhique. 
Bulletin  de  1'Academie  de  Medecine  de  Belgique,  1898,  p.  652. 


148  STUDIES  IN   IMMUNITY. 

to  the  agglutinin.  In  this  respect  he  agrees  with  Gruber's  inter- 
pretation which  we  shall  consider  later. 

Paltaufs  interpretation,  which  would  explain  the  agglutination 
of  bacteria  by  a  retraction  of  a  precipitate  (Kraus)  forming  in  the 
fluid  and  collecting  bacteria  in  its  own  clumping,  meets  with  grave 
objections.  To  begin  with  Kraus'  phenomenon  of  precipitation 
does  not  occur  constantly;  even  when  it  does  occur  the  precipitate 
is  never  abundant,  and  is  formed  so  slowly  that  it  seems  incredible 
that  it  occurs  before  the  rapid  and  energetic  bacterial  agglutination 
and  is,  therefore,  the  cause  of  it.  Moreover  no  one  has  been  able 
to  demonstrate  a  coagulum  about  agglutinated  bacteria.  Dineur, 
indeed,  has  repeatedly  attempted  to  find  one  unsuccessfully,  and 
he  very  correctly  states  that  if  there  were  such  a  coagulum  enclos- 
ing the  bacteria  we  might  expect  to  demonstrate  it  since  coagula 
take  basic  colors  as  shown  by  Nicolle. 

Certain  other  observations  may  be  noted  at  this  point.  Rabbits 
which  have  received  several  intraperitoneal  injections  of  defibri- 
nated  hen  blood  give'  a  serum  which  has  an  agglutinating  and  dis- 
solving power  for  hen  corpuscles.  This  active  serum  has  still 
another  property.  When  mixed  with  hen  serum  it  caused  a  pre- 
cipitate to  form  in  the  fluid  which  gradually  increases  and  finally 
flocks  out.  This  property  of  forming  a  precipitate  with  the  causa- 
tive serum  present  in  the  serum  of  animals  injected  with  this  serum 
was  noted  recently  for  the  first  time  by  Tchistovitch  at  the  Pasteur 
Institut.  Tchistovitch  found  that  the  serum  of  rabbits  which  had 
received  several  injections  of  eel  serum  caused  this  latter  serum  to 
become  cloudy;  he  noted  the  same  occurrence  with  the  serum  of 
rabbits  immunized  against  horse  serum  on  mixing  the  two  sera. 
As  Tchistovitch  noted,  these  precipitates  are  soluble  in  small 
amounts  of  alkali  (potassium,  sodium  or  ammonia),  as  we  also  found 
to  be  the  case  with  our  rabbit  serum  specific  for  hen  blood. 

It  would  seem  reasonable  that  these  phenomena  are  similar  to 
those  which  Kraus  noted.  The  specific  serum  from  animals  injected 
with  blood  serum  from  another  animal  causes  an  opacity  in  the 
serum  of  the  species  used  for  inoculation.  Serum  from  animals  in- 
jected with  bacterial  cultures  causes  an  opacity  in  the  culture  fluid 
in  which  the  organism  used  for  vaccination  has  grown.  The  pre- 
cipitate that  we  have  mentioned  bears  the  same  relation  to  Kraus' 


THE   MECHANISM  OF  AGGLUTINATION.  149 

precipitate  that  the  agglutinin  for  red  blood  cells  does  to  the 
agglutinin  for  bacteria.  It  may  be  shown  experimentally  that 
in  the  case  of  blood  these  precipitates  not  only  are  not  indispen- 
sable for  the  occurrence  of  strong  agglutination  but  have  no  definite 
relation  to  it.  As  we  have  already  mentioned,  the  serum  of  a  rabbit 
that  has  been  repeatedly  treated  with  hen  blood  has  the  property 
of  agglutinating  and  dissolving  hen  corpuscles  and  of  precipitating 
hen  serum.  It  also  forms  a  precipitate  with  pigeon  serum.  We 
therefore  might  expect  that  this  serum  would  also  agglutinate 
pigeon  corpuscles;  as  a  matter  of  fact  it  has  no  more  effect  on  these 
corpuscles  than  does  normal  rabbit  serum,  which  when  mixed  with 
hen  serum  remains  quite  limpid.  A  slight  agglutination,  to  be  sure, 
does  occur  either  with  the  normal  or  specific  rabbit  serum,  but  it 
is  not  so  much  as,  for  example,  the  agglutination  of  rabbit  cor- 
puscles by  normal  hen  serum,  in  which  latter  instance  also  no 
precipitate  occurs.  Guinea-pigs  which  have  been  given  several 
injections  of  defibrinated  rabbit  blood  furnish  a  serum  that  has  a 
very  intense  clumping  power  for  rabbit  corpuscles,  but  which  pro- 
duces no  clouding  with  rabbit  serum.  In  other  words  there  is  no 
necessary  parallel  between  precipitate  formation  and  an  intense 
clumping  power,  and  any  opinion  that  regards  the  formation  of 
such  precipitates  as  the  sine  qua  non  of  agglutination  would  seem 
to  be  no  longer  tenable. 

Let  us  now  consider  Gruber's  hypothesis  which  from  the  very 
beginning  has  been  extremely  open  to  criticism.  It  is  easy  enough 
to  conceive  that  a  sticky  substance  coming  from  the  covering  of 
the  bacteria  should  hold  the  micro-organisms  together,  but  it  is  not 
so  easy  to  understand  why  it  should  bring  these  organisms  together. 
No  evidence  of  the  morphological  modification  which  this  hypothe- 
sis implies  has  been  found  by  Pfeiffer  or  ourselves  on  microscopical 
examination  of  either  living  or  stained  preparations  of  bacteria  or 
red  blood  cells.  Trumpp  *  thought  that  he  did  find  an  alteration 
in  agglutinated  vibrios,  but  the  bacteria  in  which  he  noted  this 
swelling  had  been  in  fluids  containing  not  only  agglutinin,  but  alexin 
(or  lysin),  that  is,  the  bactericidal  substance  which  is  destroyed 
by  a  temperature  of  55  degrees.  This  alexin  is  very  destructive 
both  for  vibrios  and  for  red  blood  cells;  it  dissolves  the  latter  and 
*  Archiv  fur  Hygiene,  1898. 


150  STUDIES  IN   IMMUNITY. 

causes  swelling,  granular  transformation  and  often  destruction  of 
the  former.  If  he  wished  to  study  the  effect  of  the  agglutinin  alone, 
Trumpp  evidently  should  have  used  fluids  that  had  been  previously 
deprived  of  the  alexin  normally  present.*  Red  blood  corpuscles 
clumped  by  the  serum  of  an  animal  of  a  different  species  seem  to 
keep  their  normal  appearance;  they  also  remain  normal  when  sub- 
jected to  an  active  anticorpuscular  serum  that  has  been  previously 
heated  to  55  degrees  and  thus  deprived  of  its  dissolving  alexin  with- 
out losing  its  agglutinin.  It  seems,  moreover,  hardly  reasonable 
that  such  different  cells  as  bacteria  and  red  blood  cells  undergo 
the  same  modifications  when  affected  by  an  active  serum.  When 
we  deal  with  chemical  particles  such  as  milk  casein  instead  of  cells 
like  bacteria  and  red  blood  cells,  the  existence  of  a  viscous  change  is 
still  less  probable.  And  yet  a  serum  may  be  produced  that  "  agglu- 
tinates" milk,  that  is  to  say,  which  clumps  particles  of  casein. 

Is  it  reasonable  to  suppose  that  these  particles  become  sticky  or 
viscous  when  affected  by  the  active  serum?  Is  it  to  be  supposed 
that  their  sticking  together  is  due  to  such  a  viscosity?  If  we  agree 
to  this  we  must  suppose  that  particles  of  clay  in  a  homogeneous 
aqueous  suspension  are  also  covered  with  a  special  viscous  and 
sticky  coating  when  we  add  a  little  sodium  chloride  to  the  fluid 
in  which  they  are  suspended.  As  is  already  known,  the  addition 
of  salt  to  such  a  fine  clay  suspension  causes  flecks  to  form  which 
settle  to  the  bottom  of  the  tube;  this  is  a  fact  which  interests  geol- 
ogists extremely  as  a  means  of  explaining  sedimentations. 

The  existence  of  an  adhesive  substance,  which  is  the  foundation 
of  both  Gruber's  and  Dineur 's  hypotheses,  seems  to  the  latter  ob- 
server to  be  corroborated  by  a  very  significant  experiment.  Dineur 
has  noted  that  if  an  emulsion  of  bacteria  to  which  a  specific  serum 
has  been  added  is  gently  shaken,  the  clumping  of  the  bacteria 
is  very  much  increased.  Dineur  supposes  that  this  mechanical 
rolling  of  bacteria  tends  to  bring  them  together,  to  interlace  their 
cilia,  and  to  allow  the  sticky  substance  with  which  their  cilia  are 
supposedly  covered  to  bring  about  final  adhesion. 

*  The  same  criticism  may  be  made  of  the  statements  of  Roger  (Revue  ge*ne"rale 
des  Sciences,  1896)  concerning  the  modification  in  the  oidium  albicans  subjected 
to  an  active  serum.  Kraus  and  Seng  in  a  recent  article  (Wiener  klin.  Wochen- 
schrift,  1899,  No.  1)  have  offered  the  same  objections  to  the  experiments  of 
Trumpp  and  Roger. 


THE  MECHANISM  OF  AGGLUTINATION.  151 

The  fact  that  Dineur  has  reported  is  exact,  but  its  interpretation 
does  not  seem  to  be  so.  As  a  matter  of  fact,  the  favorable  effect 
of  motion  may  also  be  noted  in  the  formation  of  flecks  in  inert 
inorganic  precipitates.  If  a  drop  of  serum  is  added  to  5  or  6  c.c. 
of  a  0.7  per  cent  salt  solution  and  nitric  acid  then  added,  an  albu- 
minous opacity  is  formed  which,  if  left  alone,  clumps  very  slowly. 
But  if,  when  the  precipitate  has  been  formed,  a  small  amount  of  the 
fluid  is  poured  into  another  tube  and  this  tube  held  almost  horizon- 
tally and  gently  agitated,  the  precipitate  is  agglutinated  in  a  few 
moments  in  small  white  masses  which  float  in  a  clear  fluid.  The 
fluid  which  has  been  left  standing,  however,  remains  homogeneous 
for  a  long  time  and  the  contrast  between  the  two  is  very  striking. 
The  same  experiment  may  be  performed  with  other  albuminous 
precipitates,  as,  for  example,  with  a  precipitate  formed  by  nitric 
acid  in  whey.  The  phenomenon  to  which  Dineur  attaches  so  much 
importance  is  also  distinctly  visible  when  milk  is  agglutinated  by 
its  specific  serum.  Dineur 's  observation,  then,  instead  of  pleading 
for  Gruber's  theory  offers  a  still  further  analogy  between  the  agglu- 
tination of  bacteria  and  of  inorganic  particles.  But  there  is  another 
still  more  significant  analogy. 

We  know  that  the  collection  of  precipitates  is  frequently  con- 
trolled by  such  apparently  insignificant  causes  as  the  presence  of 
salts  in  solution  in  the  fluid.  A  clear  example  of  this  is  offered 
by  clay,  which  forms  a  very  fine  and  homogeneous  emulsion  in 
distilled  water,  but  clumps  and  falls  rapidly  in  the  tube  if 
placed  in  water  containing  sodium  chloride.  If  we  believe,  then, 
that  the  agglutination  of  bacteria  depends  on  laws  of  molecular 
adhesion,  we  might  suppose  that  salts  would  have  some  effect  on 
this  phenonenon  as  well;  and  such  indeed  proves  to  be  the  case. 
Several  24-hour  cultures  of  the  cholera  vibrio  are  suspended  in 
salt  solution  (10  c.c.  to  a  culture)  and  to  the  homogeneous  emulsion 
obtained  in  this  manner  is  added  a  powerful  agglutinating  dose  of 
cholera  serum.  The  bacteria  soon  form  flecks  which  fall  to  the 
bottom  of  the  tube.  The  tube  is  centrifugalized,  the  supernatant 
fluid  removed,  and  a  compact  mass  of  agglutinated  bacteria  left 
in  the  bottom  of  the  tube.  These  bacteria  are  then  suspended  in 
water  so  as  to  form  a  rather  thick  emulsion,  which  is  divided 
in  two  equal  parts  and  placed  in  two  separate  tubes.  To  the  first 


152  STUDIES  IN   IMMUNITY. 

tube  is  added  distilled  water  and  to  the  second  normal  salt  solution. 
After  agitation  these  tubes  are  again  centrifugalized.  It  is  found  that 
the  bacteria  in  the  tube  containing  salt  solution  go  to  the  bottom 
much  more  rapidly  than  in  the  one  containing  distilled  water. 
When  they  are  finally  deposited  the  supernatant  fluid  is  removed 
from  each  tube  and  replaced  by  a  second  amount  of  the  same  fluid, 
that  is  to  say,  salt  solution  in  one  and  distilled  water  in  the  other. 
The  bacteria  are  then  shaken  up  in  the  fluid.  It  is  found  that 
clumps  form  rapidly  in  the  tube  containing  salt  solution,  but  that  the 
bacteria  remain  indefinitely  in  suspension  in  the  tube  containing  dis- 
tilled water.  If  a  small  amount  of  the  cloudy  fluid,  say  10  c.c.,  is 
taken  from  the  second  tube  and  placed  in  a  fresh  tube  and  to  it 
is  added  0.07  grams  of  NaCl,  agglutination  reappears  and  the  deposi- 
tion of  bacteria  takes  place.* 

The  same  phenomenon  occurs  with  the  cholera  vibrio  and  a 
normal  agglutinating  serum  in  place  of  a  specific  cholera  serum. 
We  noted  three  years  ago  that  normal  horse  serum  agglutinates 
the  cholera  vibrio  and  other  bacteria  such  as  B.  typhosus,  B.  coli 
and  B.  tetani  very  markedly.  If  this  last  experiment  is  repeated 
with  normal  horse  serum  instead  of  the  specific  serum  the  same 
results  are  obtained. 

In  this  latter  case  it  is  not  necessary  to  remove  all  the  traces  of 
NaCl  by  repeated  washing.  The  salt  solution  containing  the 
clumped  bacteria  is  simply  centrifugalized  and  the  supernatant 
fluid  decanted;  the  deposition  is  then  divided  into  two  parts  and 
placed  in  two  separate  tubes ;  one  tube  is  filled  with  distilled  water 
and  the  other  with  salt  solution.  On  agitation  the  agglutination 
recurs  only  in  the  presence  of  salt.f  A  similar  experiment  with 
normal  horse  serum  and  B.  typhosus  gave  the  same  result. 

*  It  must  be  noted  that  this."reagglutination"  of  bacteria  on  the  addition  of 
salt  to  distilled  water  does  not  occur  quite  so  rapidly  as  in  the  tube  in  which  the 
bacteria  have  remained  in  contact  with  salt  solution,  especially  when  the  contact 
with  distilled  water  has  been  prolonged.  It  is  probable  that  the  micro-organisms 
must  retain  a  certain  amount  of  salt  in  order  to  agglutinate  well. 

t  The  presence  of  an  agglutinating  power  in  serum  has  doubtless  vitiated  many 
researches  on  the  bacterial  power  of  body  fluids.  Many  observers,  indeed,  have 
used  the  method  of  inoculating  at  intervals  small  amounts  of  a  mixture  of  serum 
and  bacteria  in  gelatin  in  order  to  determine  the  destructive  power  of  the  serum. 
It  is  quite  possible  that  the  serum  in  question,  when  the  amount  of  bacteria  is  small, 
may  so  clump  them  that  each  clump  of  bacteria  will  give  rise  to  a  single  colony 


THE  MECHANISM   OF  AGGLUTINATION.  153 

The  same  results  are  also  obtained  if,  instead  of  bacteria,  Kraus' 
precipitate,  obtained  by  mixing  cholera  serum  with  an  old  filtered 
culture  of  the  vibrios,  is  used.  This  precipitate  is  treated  exactly 
as  described  for  bacteria,  and  it  is  found  that  it  clumps  very  much 
more  markedly  in  fluids  containing  salt  than  in  distilled  water. 

It  may  be  worth  while  to  give  the  results  obtained  by  the  same 
technique  on  the  agglutination  of  a  fine  emulsion  of  potter's  clay 
in  distilled  water  after  filtering  through  paper.  Tubes  which  do  not 
contain  salt  remain  opaque  for  days,  whereas  in  tubes  containing 
0.7  per  cent  salt  solution  there  is  a  very  distinct  agglutination  and 
a  rapid  deposition.  The  resemblance  between  floating  clumps  of 
agglutinated  bacteria  and  the  whitish  flecks  of  clay  suspended  in 
salt  solution  and  falling  slowly  to  the  bottom,  is  very  striking. 

These  experiments  on  the  absence  of  agglutination  in  distilled 
water  are  very  strongly  confirmatory  of  the  idea  that  the  agglutinin 
acts  by  producing  on  isolated  elements  such  changes  in  their  proper- 
ties  of  molecular  adhesion  as  are  shown  by  particles  of  clay.  In  each 
case  the  adding  of  salt  suffices  to  produce  the  physical  phenomenon 
of  agglutination  which,  without  it,  is  impossible.  This,  in  our 
opinion,  is  a  confirmation  of  an  hypothesis  that  we  formerly  offered 
and  have  just  recalled. 

*** 

If  we  conceive  of  the  phenomenon  of  agglutination  in  this  way, 
interesting  generalizations  may  be  drawn,  particularly  in  the  light 
of  the  explanation  that  Duclaux  has  proposed  for  coagulation. 
What,  indeed,  is  agglutination?  It  is  the  union  into  masses  of  organ- 
ized scattered  particles,  by  some  peculiar  influence  that  changes 

only.    This  technical  error  is  naturally  of  great  importance  in  researches  on  easily 
agglutinable  bacteria  like  B.  typhosus. 

It  is  certain  that  real  bactericidal  properties  have  been  presumed  to  exist 
when  agglutinins  alone  were  present.  It  is  also  probable  that  at  times  certain 
properties  of  the  agglutinins  have  been  attributed  to  the  alexins.  For  example, 
Buchner  states  that  alexin  loses  its  activity,  to  a  large  extent  at  least,  when  mixed 
with  distilled  water.  The  presence  of  distilled  water  might  apparently  diminish 
the  bactericidal  property  of  a  serum  by  weakening  its  agglutinating  power  and 
consequently  by  increasing  the  number  of  colonies  that  grow  on  gelatin.  We 
have  found  that  alexin  acts  very  well  in  a  medium  with  very  little  salt;  vibrios 
treated  with  preventive  serum  and  then  washed  and  suspended  in  fifteen  parts 
of  distilled  water  may  show  granular  transformation  when  one  part  of  normal 
serum  is  added  to  these  fifteen  parts  of  emulsion,  without  any  agglutination. 


154  STUDIES  IN   IMMUNITY. 

the  properties  of  molecular  adhesion.  What  is  coagulation  accord- 
ing to  Duclaux?  It  is  the  uniting  in  groups  of  particles  which  may 
have  been  so  finely  divided  as  to  appear  in  solution,  by  some  peculiar 
influence  which  modifies  the  molecular  relations  between  the  par- 
ticles and  the  fluid.  Before  the  intervention  of  this  influence  the 
liquid  remains  homogeneous,  but  as  a  result  of  it  "the  state  of  equi- 
librium between  gravity  and  molecular  forces  is  disturbed,  either 
because  the  adhesion  between  the  fluid  and  the  solid  has  diminished, 
or,  more  probably,  because  the  attraction  between  the  particles  of 
the  solid  is  increased  so  that  they  unite  into  more  or  less  voluminous 
collections  which  become  visible  to  the  naked  eye  and  are  precipi- 
tated."* 

On  account  of  these  changes  of  molecular  adhesion,  particles, 
the  chemical  nature  of  which  may  differ  greatly  in  the  various 
instances  and  which  are  often  so  small  as  not  to  be  microscopically 
visible,  collect  into  masses  which  are  still  invisible  to  the  naked 
eye,  but  which,  by  progressive  clumping,  gradually  increase  in  size 
and  render  the  fluid  opaque,  until  by  molecular  condensation  they 
form  more  and  more  voluminous  masses. 

It  is  not  necessary  to  follow  the  systematic  way  in  which  Duclaux 
has  elaborated  this  idea,  nor  to  indicate  how  simplifying  this  con- 
ception is,  combining  as  it  does  facts  that  were  so  separated  as  to 
appear  unrelated.  Such  a  conception  connects  agglutination 
with  the  phenomenon  of  coagulation,  as  he  conceived  of  it.  The 
agglutination  of  bacteria  is  due  to  a  change  in  molecular  adhesion 
between  the  bodies  of  the  bacilli  and  the  surrounding  fluid.  As 
Duclaux  expresses  it  this  phenomenon  "as  a  whole  and  in  detail 
recalls  our  observations  and  description  given  in  the  chapter  on  the 
phenomenon  of  coagulation."! 

Therefore,  if,  as  Duclaux  affirms,  we  have  the  right  to  regard 
agglutination  as  a  phenomenon  of  coagulation  and  if  we  are 
authorized  in  giving  henceforth  the  active  substance  in  serum  the 
more  suggestive  name  of  "coagulin"  instead  of  agglutinin,  which 
latter  term  simply  indicates  its  activity  without  any  reference  to 
its  relations  or  cause,  we  may  suppose  that  the  animal  body,  owing 
to  its  functional  plasticity  and  the  multiplicity  of  its  resources,  would 
be  able  to  elaborate,  when  necessary,  active  clumping  principles, 

:!  Duclaux,  Trait£  de  microbiologie,  vol.  2,  p.  263.        t  Duclaux,  Ibid,  p.  706. 


THE  MECHANISM  OF  AGGLUTINATION.  155 

not  only  against  organized  cells  but  against  such  chemical  sub- 
stances as  have  been  recognized  as  coagulable. 

Experiment,  indeed,  justifies  this  supposition.  If  rabbits  are 
given  several  successive  intraperitoneal  injections  of  milk,  pre- 
viously heated  to  65  degrees  to  sterilize  it  partially,  after  a  proper 
interval  they  give  a  serum  that  has  specific  properties  against  milk. 

A  certain  amount  of  this  serum  (for  example  3  c.c.),  is  placed  in 
a  tube  and  the  same  amount  of  normal  rabbit  serum  is  placed  in 
another  tube.  To  each  of  these  tubes  milk  is  added  (10  to  15 
drops  for  example).  The  tube  containing  normal  serum  remains 
opalescent  and  homogeneous.  In  the  tube  containing  active 
serum  small  particles  rapidly  appear  that  soon  increase  in  size 
and  form  thick  flecks.  The  fluid  then  becomes  separated  into  two 
parts,  one  of  which  is  quite  limpid  and  the  other  of  which  con- 
tains clumped  masses  which  generally  fall  to  the  bottom  of  the 
tube,  leaving  the  clear  fluid  above.  This  sedimentation  goes  on 
better  if  milk  that  contains  little  fat  or,  better  still,  milk  that  has 
been  passed  two  or  three  times  through  filter  paper  and  so  deprived 
of  part  of  its  fat' globules,  is  used.*  If  milk  containing  a  good  deal 
of  fat  is  used,  the  clumps  may  float  to  the  top  carried  up  by  the 
fat  corpuscles  that  they  have  enclosed. 

If  these  mixtures  of  milk  with  normal  serum  or  with  "lactoserum  " 
are  passed  through  a  filter  paper,  the  latter  mixture  filters  quite 
clear  without  any  of  the  whitish  opacity  which  milk  produces;  the 
mixture  containing  normal  serum  remains  cloudy  after  filtration. 

Microscopical  examinations  of  these  mixtures  of  milk  with  sera 
show  that  the  lactoserum  causes  the  formation  of  abundant  granu- 
lar masses  that  do  not  occur  in  mixtures  with  normal  serum.  This 
granular  precipitate  resembles  the  clots  of  casein  formed  by  rennin.f 

*  The  experiment  is  more  striking  if  filtered  milk  of  this  sort  is  used.  Such 
milk  does  not  stick  to  glass  that  it  touches,  and  renders  the  fluid  less  opaque,  so 
that  the  agglutination  may  be  more  readily  estimated. 

t  We  do  not  wish  at  all  to  assert  that  the  agglutinin  of  lactoserum  is  identical  in 
action  with  rennin.  There  are  distinct  differences  between  these  two  substances. 
The  action  of  the  agglutinin  is  much  less  dependent  on  a  suitable  temperature  than 
is  rennin;  as  a  matter  of  fact,  it  acts  at  a  temperature  so  low  that  rennin  is  almost 
inactive.  Nor  does  our  serum  have  the  property  of  clotting  relatively  enormous 
quantities  of  casein,  as  does  rennin.  Moreover,  in  a  mixture  containing  a  large 
amount  of  normal  serum  and  a  small  amount  of  milk,  rennin  has  slightly,  any 
effect,  whereas  under  these  conditions  agglutination  appears  on  addition  of 
lactoserum. 


156  STUDIES  IN   IMMUNITY. 

Certain  of  these  clumps  are  composed  entirely  of  a  fine  granular 
precipitate,  and  others  enclose  a  large  number  of  fat  globules  in 
their  midst.  If  a  little  more  milk  than  can  be  agglutinated  is  added 
to  lactoserum,  there  is  nevertheless  an  abundant  deposit  of  the 
agglutinated  substance  formed.  After  the  deposition  of  all  the 
clumps,  even  to  the  smallest  ones,  the  supernatant  fluid  is  found  to 
be  quite  limpid.  When  taken  off  and  mixed  with  normal  serum 
this  supernatant  fluid  gives  no  opacity.  If  added  to  lactoserum, 
however,  it  causes  a  slight  cloud  which  soon  increases  and 
is  followed  by  flecks  which  form  a  considerable  deposit.  In  other 
words,  the  lactoserum  has  clumped  casein  that  was  in  so  fine  a  state 
of  division  that  it  did  not  render  the  liquid  turbid,  and  that  had 
escaped  the  agglutinating  action  of  the  first  insufficient  dose  of 
active  serum. 

A  similar  experiment  may  be  performed  in  the  following  manner: 
as  we  have  already  stated,  normal  serum  containing  milk  (for 
example,  4  c.c.  of  serum  to  ten  drops  of  milk)  passes  through  filter 
paper  as  an  opaque  fluid.  If  the  filtration  is  repeated  a  number  of 
times  through  the  same  paper,  a  much  clearer  fluid,  which  is  only 
almost  imperceptibly  opalescent,  is  finally  obtained.  On  micro- 
scopic examination  this  fluid  contains  very  few  fat  globules  and 
nothing  else.  If  a  small  amount  of  this  is  mixed  with  equal  parts 
of  normal  serum,  nothing  happens.  When  mixed  with  lactoserum 
in  equal  parts  the  fluid  that  was  at  first  transparent  immediately 
becomes  turbid,  and  rather  voluminous  white  masses  of  casein 
form,  which,  microscopically,  are  granular  clumps,  identical  with 
those  formed  by  rennin. 

Similar  precipitates  may  also  be  formed  with  whey  produced 
by  adding  rennin  to  milk.  This  whey  when  filtered  is  faintly 
opalescent  and  causes  no  turbidity  with  normal  serum;  when  added 
to  lactoserum  it  causes  a  very  distinct  turbidity  which  soon  settles 
down  in  the  form  of  flecks.  It  is  well  known  that  whey  still  con- 
tains casein  which  has  escaped  the  effect  of  the  rennin,  but  which 
gives  a  voluminous  precipitate  on  addition  of  an  acid. 

*** 

Are  there  no  analogies  to  be  drawn  between  the  appearance  of 
flaky  precipitates  in  limpid  fluids  containing  sufficient  casein  to 
render  them  faintly  opalescent  when  lactoserum  is  added,  and  the 


THE  MECHANISM  OF   AGGLUTINATION.  %    157 

precipitates  produced  on  mixing  two  sera  under  the  conditions 
already  described?  Our  specific  serum  from  a  rabbit  injected  with 
hen  blood  causes  an  abundant  precipitate  with  hen  serum.  May 
we  not  assume  in  this  case,  too,  that  the  active  serum  collects  molec- 
ular groups  which  have  previously  remained  scattered  and  disso- 
ciated to  such  an  extent  that  they  did  not  cloud  the  limpid  fluid? 
The  precipitate  produced  in  such  mixtures  of  sera  would  seem  to 
be  caused  by  a  phenomenon  of  agglutination,*  or,  if  preferred,  of 
coagulation,  for  we  are  at  a  loss  to  know  which  of  the  two  terms 
to  use. 

May  we  not  also  draw  an  analogy  between  the  appearance  of  these 
precipitates  and  the  agglutination  of  bacteria  or  of  red  blood  cells 
which  is  nothing  more  than  the  collecting  into  voluminous  masses 
of  separate  defined  cells?  The  only  point  of  difference  between 
the  phenomena  in  question  is  that  in  certain  of  them  the  aggluti- 
nable  particles  are  so  small  and  separate  that  before  being  collected 
they  fail  to  affect  the  limpidity  of  the  liquid ;  in  certain  other  cases, 
as  with  bacteria  or  corpuscles,  they  are  sufficiently  large  to  pro- 
duce a  visible  cloud  before  being  clumped. 

But  this  variation  in  size  of  the  particles  concerned  is  only  an 
accessory  fact  which,  in  our  opinion,  certainly  does  not  affect  the 
essentials  of  the  phenomena  themselves.  Such  a  distinction  is  only 
secondary  and  cannot  in  the  least  affect  the  conclusion  that  there  is 
no  fundamental  difference  between  the  phenomena  of  agglutination  and 
of  coagulation.  For  example,  the  coagulation  of  clay  is  closely 
allied  to  the  coagulation  of  milk,  according  to  Duclaux's  ideas. 
It  is,  moreover,  closely  related  to  the  agglutination  of  bacteria,  as 
is  shown  by  the  effect  of  sodium  chloride;  and  further,  the  agglu- 
tination of  bacteria  resembles  the  coagulation  of  milk,  as  we  learn 
from  the  experiment  with  agglutinating  lactoserum,  which  produces 
coagulation  similar  to  that  caused  by  a  mixture  of  normal  serum  and 

*  The  following  fact  corroborates  this  point  of  view.  The  specific  property 
of  the  active  rabbit  serum  that  produces  a  precipitate  with  hen  serum  is  weakened 
on  heating  to  65  degrees  for  half  an  hour;  it  is  destroyed  on  heating  for  the  same 
length  of  time  to  70  degrees.  On  heating  to  65  degrees  or,  still  more  so,  by  heating 
to  70  degrees  the  serum  also  loses  to  a  great  extent  its  agglutinating  power  for 
hen  corpuscles.  In  other  words,  the  precipitating  substance  is  affected  by  heat 
in  the  same  way  as  is  the  agglutinin.  The  precipitable  substance  of  hen  serum, 
on  the  contrary,  resists  heating  to  75  degrees  for  half  an  hour,  as  is  shown  by  its 
forming  a  precipitate  when  added  to  active  rabbit  serum. 


158  STUDIES  IN   IMMUNITY. 

antiserum.  This  specific  precipitating  serum,  moreover,  is  pro- 
duced by  treating  animals  much  in  the  same  way  as  to  produce 
agglutinating  sera  for  bacteria.  There  are  numerous  evidences  of 
similarity  between  these  different  phenomena,  so  that  one  is  forced 
to  accept  a  single  general  explanation  for  them  all,  and  to  attribute 
the  occurrence  of  agglutination  to  changes  in  molecular  adhesion. 

If  we  were  to  choose  the  specific  agglutination  of  bacteria  as  an 
example  of  these  phenomena  we  might  say  that  the  agglutinin  which 
unites  with  the  bacteria  acts  by  modifying  the  relations  of  molecular 
attraction  both  between  the  individual  bacterial  particles  and 
between  these  particles  and  the  surrounding  fluid.  The  agglutinin 
affects  only  a  certain  definite  bacterial  species.  During  the  first 
period  of  agglutination  the  individual  constitution  of  the  bacteria 
in  question  is  much  in  evidence.  It  is,  perhaps,  essential  that  bac- 
teria, in  order  to  be  affected  by  an  agglutinin,  should  be  suffi- 
ciently intact,  as  would  seem  indicated  by  certain  of  Malvoz's* 
experiments. 

But  as  soon  as  the  change  in  molecular  adhesion  has  been  produced 
the  bacteria  collect  as  do  inorganic  particles.  It  is  not  necessary  to 
imagine  that  their  structure  has  anything  to  do  with  it ;  nor  to  think 
that  the  bacteria  must  stick  to  one  another  as  a  label  sticks  to  a 
bottle,  by  means  of  some  peculiar  adhesive  substance  which  covers 
the  cilia,  or  owing  to  a  swollen  outer  membrane.  This  phenomenon 
of  the  collection  of  particles  by  means  of  some  influence  which 
changes  their  molecular  attraction  should  by  definition  be  placed 
among  the  phenomena  of  coagulation  as  Duclaux  has  described 
them. 

From  this  standpoint  then  the  phenomenon  of  agglutination  is 
divided  into  two  distinct  phases.  In  the  first  phase  the  scattered 
bacteria  are  affected  by  the  agglutinin  and  absorb  it.  This  causes 
modifications  in  their  properties  of  molecular  adhesion.  The 
existence  of  these  modifications  during  the  second  phase  brings 
about  agglutination  properly  speaking. 

This  division  into  two  periods  is  neither  artificial  nor  imaginary. 
These  two  phases  may  indeed  be  separated  and  the  first  be  brought  about 
without  the  second.  The  experiment  already  mentioned  that  shows 

*  Recherches  sur  I'agglutination  du  bacille  typhique.  Annales  de  1'Institut 
Pasteur,  July,  1897 


THE  MECHANISM  OF  AGGLUTINATION.  159 

the  function  of  sodium  chloride  does  this  very  thing.  The  bac- 
teria that  we  washed  and  suspended  in  distilled  water  had  been 
affected  by  the  agglutinin.  They  are  immobilized  and  are  ready 
to  be  agglutinated  energetically.*  But  to  bring  about  the  second 
phase  of  the  phenomenon  or  agglutination,  properly  speaking,  a  little 
salt  must  be  added  to  the  emulsion. 

As  far  as  Kraus'  phenomenon  is  concerned  we  are  as  yet  in  no 
position  to  interpret  it.  It  is  by  no  means  demonstrated  that 
Kraus'  precipitates  have  anything  to  do  with  the  real  agglutination 
of  bacteria;  it  may  be  that  this  precipitate  is  similar  to  that  obtained 
in  a  mixture  of  defibrinated  hen  blood  and  specific  rabbit  serum 
(that  is,  serum  from  a  rabbit  injected  with  hen  blood),  which  appar- 
ently has  no  relation  to  the  agglutination  of  the  corpuscles  them- 
selves. But  if  Kraus'  precipitate  is  formed  from  the  agglutinable 
substance  of  bacteria  it  seems  to  us  that  we  must  compare  it,  from 
the  standpoint  of  its  formation,  with  the  casein  precipitates  which 
lactoserum  produces  with  whey,  in  which  instance  the  substance 
is  so  finely  divided  as  not  to  disturb  the  limpidity  of  the  fluid.  In 
other  words  this  precipitation  would  resemble  the  agglutination 
of  bacterial  substances  in  a  finely  divided  state. 


* 
*  * 


The  idea  that  the  agglutination  of  cells,  corpuscles,  bacteria  or 
non-differentiated  particles  like  casein  has  the  characteristics  of  a 
phenomenon  of  coagulation  might  suggest  certain  observations  on 
the  significance  of  the  active  properties  of  serum.  Let  us  enumerate 
briefly,  without  repeating  the  observations  that  we  have  formerly 
made,  the  essential  properties  which  we  have  found  in  specific  sera 
by  experiments  performed  in  vitro.  To  be  more  exact  we  shall 
consider  the  two  sera  which  we  used  as  typical,  that  is  cholera  serum, 
and  a  serum  active  against  rabbit  corpuscles  obtained  by  injecting 
guinea-pigs  with  rabbit  blood.  These  sera,  as  we  have  recently 
pointed  out,  have  similar  properties  which  are  as  follows : 

1.  Both  sera  agglutinate  cellular  elements  and  suppress  their 
motility  if  they  have  any. 

2.  When  either  the  vibrios  or  the  corpuscles  have  been  in  con- 
tact with  their  respective  serum  they  are  more  susceptible  to  the 

*  We  may  also  add  that  they  have  become  very  susceptible  to  the  effect  of 
alexin. 


100  STUDIES  IN   IMMUNITY. 

destructive  effect  of  alexin.  This  alexin  or  lysin  is  the  bactericidal 
or  globulicidal  substance  that  is  destroyed  at  55  degrees,  and  affects 
certain  delicate  cells  as  a  sort  of  digesting  diastase.  The  specific 
properties  of  these  sera  resist  heating  to  55  degrees  or  60  degrees. 

3.  The  sera  when  fresh  contain  alexin;  and  it  is  due  to  the 
presence  of  this  substance  that  they  are  able  to  alter  profoundly 
or  to  cause  partial  dissolution  of  those  cells  which  they  sensitize. 

If  we  were  to  enumerate  the  really  essential  properties  of  these 
sera  we  might  eliminate  the  third,  namely  the  possession  of  alexin, 
as  this  substance  is  also  present  in  the  serum  of  normal  animals. 
After  destroying  this  alexin  in  the  specific  serum  by  heating  it  to 
55  degrees  and  so  removing  its  bactericidal  or  globulicidal  power, 
the  power  may  be  Testored  by  the  addition  of  a  small  amount  of 
normal  serum  which  contains  alexin.* 

There  remain  then  two  other  properties,  which,  to  be  sure,  occur 
in  normal  sera  to  a  slight  extent,  but  may  be  considered  as  charac- 
teristic of  the  serum  of  vaccinated  animals. 

We  shall  not  consider  here  the  question  as  to  whether  these  two 
properties  are  due  to  two  distinct  substances  or  are  to  be  attributed 
to  the  action  of  one  and  the  same  substance,  reserving  such  dis- 
cussion for  another  time.  In  all  events  the  most  remarkable  of 
these  two  properties  is  the  sensitizing  of  cells  to  the  action  of  the 
alexin.  When  we  say  that  there  exists  in  specific  sera  a  sensitizing 
substance  (substance  sensibilisatrice)  it  implies  that  these  sera  act 
directly  on  the  cells.  Indeed  this  sensitizing  substance  has  a  par- 
ticular predilection  for  fixing  itself  on  those  cells  which  it  affects. 

When  cholera  vibrios  are  placed  in  a  suitable  amount  of  fluid 
containing  cholera  serum  they  absorb  its  active  principles.  After 
centrifugalizing  and  decanting  the  clear  supernatant  fluid  is  found 
to  have  lost  both  its  agglutinating  power  and  its  power  of  sen- 
sitizing new  bacteria  to  the  action  of  the  alexin.  In  other  words, 
new  vibrios,  when  placed  in  contact  with  this  fluid,  are  not  immo- 
bilized or  agglutinated  and  may  be  injected  into  the  peritoneal  cavity 
of  a  guinea-pig  or  mixed  with  normal  guinea-pig  serum  in  vitro  with- 
out showing  granular  transformation.  The  same  phenomenon  of 
absorption  or  fixation  occurs  if  the  vibrios  are  cultivated  in  bouillon 

*  The  details  of  these  experiments  for  the  cholera  vibrio  will  be  found  in  the 
article  beginning  page  50,  and  for  the  red  blood  cells  in  the  article  on  page  134. 


THE  MECHANISM   OF  AGGLUTINATION.  161 

to  which  a  moderate  amount  of  cholera  serum  has  been  added. 
The  bacteria  in  their  development  remove  all  the  particular  proper- 
ties from  the  fluid*  The  same  facts  hold  for  sera  -active  against 
red  blood  cells.  When  red  blood  cells  are  placed  in  contact  with 
their  specific  serum  they  absorb  both  the  agglutinating  substance 
and  the  sensitizing  substancef.  The  serum  separated  by  centrifu- 
galizing  such  a  mixture  is  inactive  for  fresh  corpuscles.  It  may 
also  be  added  that  the  agglutinins  of  normal  sera  are  likewise  ab- 
sorbed by  corpuscles  or  bacteria.  £  This  all  indicates  that  animals 
during  vaccination  are  to  be  distinguished,  not  by  the  elaboration 
of  large  amounts  of  the  "dissolving  diastase"  or  alexin,  but  by 
formation  of  those  substances  which  favor  the  action  of  these  diastases, 
that  is,  of  principles  which  unite  with  the  cells  and  sensitize  them 
to  the  effect  of  this  alexin. 

*  This  result  completely  contradicts  the  facts  observed  by  Pfeiffer,  who  per- 
formed this  last  experiment.  (Centralblatt  fur  Bakt.,  1896.)  Pfeiffer  found 
that  the  agglutinin  was  absorbed  under  these  conditions,  but  stated  that  the  fluid 
in  which  bacteria  had  grown  still  retained  the  property  of  causing  a  granular 
transformation  when  injected  with  new  vibrios  into  the  peritoneal  cavity  of  a 
normal  guinea-pig.  Pfeiffer's  conclusion  that  active  sera  do  not  act  on  bacteria 
in  the  same  manner  in  vitro  as  they  do  in  the  peritoneal  cavity  is  erroneous.  The 
effect  in  vivo  and  in  vitro  is  precisely  the  same.  Experiment  shows  us  that  the 
smallest  amount  of  cholera  serum  necessary  to  cause  granular  transformation  of  a 
given  dose  of  vibrios  is  the  same  whether  the  transformation  takes  place  by  means  of 
the  alexin  in  the  peritoneal  cavity,  or  in  vitro  on  the  addition  of  the  alexin  of  normal 
serum.  This  minimal  destructive  dose  for  vibrios  is  very  similar  to  the  minimal 
agglutinating  dose,  as  we  shall  consider  presently. 

f  It  must  be  noted  that  Ehrlich  (Collected  Studies  on  Immunity,  Ehrlich- 
Bolduan,  Wiley  &  Co.,  page  1)  has  recently  noted  that  hemolytic  serum  exhausted 
by  contact  with  its  specific  corpuscles  no  longer  forms  a  dissolving  mixture  for 
new  corpuscles  on  the  addition  of  normal  serum. 

t  We  should  like  to  mention  briefly  a  curious  experiment  which,  strictly  speak- 
ing, is  rather  irrelevant.  If  a  given  dose  of  normal  serum  with  strong  aggluti- 
nating property  for  cholera  vibrios  (normal  horse  serum)  is  placed  with  these 
vibrios,  agglutination  takes  place.  If  the  mixture  is  then  centrifugalized  and  the 
clear  supernatant  fluid  taken  off,  it  is  found  that  it  no  longer  agglutinates  cholera 
vibrios,  but  still  does  agglutinate  the  typhoid  bacillus.  Conversely,  normal  'horse 
serum  mixed  with  typhoid  bacilli  leaves  a  supernatant  fluid  after  agglutination 
and  centrifugalization  that  no  longer  agglutinates  the  typhoid  bacillus,  but  still 
agglutinates  the  cholera  vibrio.  It  seems  certain,  then,  that  there  are  two  distinct 
agglutinins,  one  for  each  of  these  micro-organisms,  in  the  same  serum.  Such 
experiments  may  one  day  throw  light  on  the  obscure  question  of  the  origin  of  the 
specificity  of  active  properties  in  serum.  It  is  quite  conceivable  that  vaccination 
with  a  given  bacterium  may  cause  the  production  of  a  large  amount  of  an  agglu- 
tinin which  has  already  existed  in  small  amounts. 


162     .  STUDIES  IN   IMMUNITY. 

It  is  owing  to  the  presence  of  these  facilitating  substances  that 
the  sera  of  vaccinated  animals  can  produce  marked  evidence  of 
digestion  if  the  cells  affected  are  not  too  resistant,  as,  unfortunately, 
is  the  case  with  many  bacteria.*  We  may  consider  such  sera,  then, 
as  analogous  to  digestive  secretions. 

This  analogy  between  the  properties  of  active  sera  and  digestive 
secretions  is  still  more  evident  when  we  consider  that  the  active 
substances  in  serum  arise  in  the  digestive  cells  that  Metchnikoff 
demonstrated;  the  function  in  immunity  of  such  cells  is  very  im- 
portant and,  in  the  course  of  evolution,  they  become  identified  with 
those  ameboid  cells  which,  in  simple  organisms,  are  able  to  assure 
the  nutrition  of  the  individual,  owing  to  their  intracellular  digestive 
functions.  It  is  such  cells  that,  as  Metchnikoff  has  found,  take 
up  the  function  of  digestion  in  slightly  differentiated  species.  What 
is  more,  such  ameboid  cells  represent  the  origin  of  our  digestive 
apparatus,  for,  as  Metchnikoff  has  shown,  digestion,  which  at  first 
is  only  intracellular,  becomes,  in  the  course  of  evolution,  extracellu- 
lar, as  these  cells  acquire  the  property  of  excreting  their  dissolving 
juices.  The  source  of  the  agglutinins  and  the  sensitizing  substances 
is  not,  to  be  sure,  well  defined,  but,  as  far  as  the  alexin  or  dissolving 
principle  is  concerned,  we  know  from  numerous  observations  that 
it  is  of  leucocytic  origin. 

In  the  present  state  of  our  knowledge  of  this  as  yet  obscure 
subject  we  do  not  wish  to  state  too  dogmatically  such  facts  as  occur 
to  us  as  correlated.  There  are  at  least  important  characteristics 
which  suggest  the  relation  of  active  sera  to  digestive  juices,  and 
the  analogy  would  be  explicable  if,  as  numerous  facts  and  supposi- 
tions render  it  probable,  we  were  to  attribute  the  elaboration  of 
these  active  substances  to  the  phagocytes,  in  other  words,  to  that 
group  of  cells  endowed  with  digestive  properties  which  are  retained 
during  evolution. 

If  future  facts  point  to  the  same  conclusions,  we  shall  finally 
regard  immunity,  not  only  from  the  biological  standpoint,  as  we  do 
at  the  present  moment,  but  also  from  the  chemical  standpoint,  as  an 
instance  of  the  physiology  of  digestion. 

*  As  we  know,  the  typhoid  bacillus  and  the  colon  bacillus  show  only  slight 
granular  transformation  in  the  presence  of  an  active  serum.  Many  other  bacteria 
are  still  less  susceptible  to  bactericidal  properties. 


THE   MECHANISM  OF   AGGLUTINATION.  163 

CONCLUSIONS. 

I.  Theories  that  explain  bacterial  agglutination  by  a  swelling 
and  viscosity  either  of  the  membranes  or  cilia  of  the  bacteria  meet 
with  numerous  objections  and  do  not  serve  to  explain  all  the  phe- 
nomena of  agglutination.     The  theory  that  regards  the  forma- 
tion of  a  precipitate  in  the  fluid  as  the  cause  of  agglutination  is  open 
to  the  same  objections. 

II.  Agglutination  may  affect  such  different  elements  as  red 
blood  corpuscles,  bacteria,  and  casein.     In  all  these  instances  of 
agglutination  by  serum  the  same  explanation  must  be  accepted. 

III.  We  may  conclude  that  the  agglutinins  by  uniting  with  the 
agglutinable  substances  lead  to  changes  in  molecular  attraction 
between  the  elements  affected,  either  as  among  themselves  or  as 
between  them  and  the  surrounding  fluid.     The  entire  phenomenon 
of  agglutination  should  be  divided  into  two  phases,  of  which  the 
first  may  be  experimentally  produced  without  the  second.    The 
first  is  a  period  during  which  the  isolated  elements  are  affected  by 
the  agglutinin,  and  the  second  a  period  of  agglutination,  properly 
speaking.     The  individuality  of  the  elements  affected  counts  only 
in  the  first  phase.     During  the  second  phase,  cells  in  obedience  to 
molecular  attraction  show,  in  their  agglutination,  only  such  pecul- 
iarities as  occur  in  the  clumping  of  mineral  particles. 

IV.  The  phenomena  of  agglutination  resemble  the  phenomena 
of  coagulation  very  closely. 

V.  The   phenomena    of  true    agglutination    may    be   brought 
about  in  limpid  fluids  in  which  the  particles  are  extremely  finely 
divided. 

VI.  Active  sera  may  be  compared  with  digestive  juices  in  respect 
to  their  coagulating  and  dissolving  properties.     It  would  seem  as 
if  immunity  would  come  to  be  regarded  more  and  more  from  a 
chemical  standpoint  as  an  instance  of  the  physiology  of  digestion. 

VII.  As  was  previously  stated  in  an  article  on  sera  active  against 
blood   corpuscles,   the  production   of  bacteriolytic  substances  in 
animals  during  the  course  of  vaccination  cannot  be  regarded  tele- 
ologically.     The  animal  body  does  not  form  these  harmful  sub- 


164  STUDIES   IN   IMMUNITY. 

stances  for  the  purpose  of  defending  itself,  but  simply  puts  into 
action  preexisting  functional  capacities  that,  under  proper  condi- 
tions, might  act  on  such  innocuous  substances  as  red  blood  cells  or 
milk  casein,  as  well  as  bacteria.  The  special  properties  that  are 
found  in  the  sera  of  vaccinated  animals  are  present  in  a  primitive 
form  in  normal  sera.  This  fact  probably  has  a  distinct  bearing  on 
the  specific  nature  of  these  substances  in  immune  sera. 


VIII.    THE  AGGLUTINATION  AND  DISSOLUTION  OF  RED 
BLOOD  CELLS  BY  SERUM.*     (SECOND  MEMOIR.) 

BY  DR.   JULES   BORDET. 

In  the  present  article  we  shall  offer  certain  facts  in  addition  to 
those  recently  published.!  In  our  preceding  memoir  we  enum- 
erated the  properties  found  in  serum  of  guinea-pigs  that  have 
received  several  injections  of  defibrinated  rabbit  blood  and  we  in- 
sisted on  the  close  analogy  between  the  properties  of  such  a  serum 
and  those  of  an  antimicrobial  serum  like  cholera  serum. 

We  shall  now  consider,  first,  whether  the  cellulicidal  property  is 
the  sole  characteristic  of  antihematic  sera  or  whether  there  are, 
in  addition,  certain  antitoxic  properties.  We  shall  then  endeavor 
to  draw  a  still  further  comparison  between  antihematic  sera  and 
antimicrobial  sera.  For  example,  we  shall  consider  whether  an 
antihematic  serum  injected  into  a  normal  animal  endows  its  fluids 
with  a  cellulicidal  property,  for  we  know  that  the  injection  of  cholera 
serum  into  an  animal  gives  rise  in  the  serum  of  the  recipient  to  an 
intense  bactericidal  power. 

A  comparison  must  be  drawn  between  the  important  properties 
of  these  different  sera.  It  does  not  suffice  simply  to  compare  anti- 
hematic  sera  with  antimicrobial  sera.  We  must  still  further  con- 
sider whether  normal  sera  and  specific  sera  have  any  characters 
in  common,  and  whether  the  active  properties  which  develop  and 
are  specialized  as  a  result  of  immunizing  injections  are  present  in 
a  primitive  form  in  normal  animals.  It  is  already  known  that  the 
majority  of  normal  sera  have  a  faculty  of  agglutinating  and  destroy- 
ing alien  red  blood  cells  and  also  certain  bacteria  in  some  degree. 
These  evident  analogies  must  be  carefully  outlined. 

*  Agglutination  et  dissolution  des  globules  rouges  par  le  se>um.     Annales  de 
Tlnstitut  Pasteur,  1899,  XIII,  p.  273. 
t  See  article,  page  134. 


165 


166  STUDIES  IN  IMMUNITY. 


THE  PROPERTIES  OF  ANTIHEMATIC  SERA. 

The  active  serum  used  in  our  previous  work  was  furnished  by 
guinea-pigs  that  had  received  several  injections  of  defibrinated 
rabbit  blood.  This  serum  has  a  harmful  effect  on  rabbit  corpuscles 
in  that  it  agglutinates  them  and  brings  about  their  dissolution. 
In  other  words,  it  attacks  the  identical  cells  which  have  been  used 
to  inject  the  animals  that  have  furnished  the  serum. 

But  it  might  well  be  imagined  that  such  a  serum  also  possesses 
" defensive  properties"  in  addition  to  ''attacking  properties." 

Let  us  consider,  for  example,  the  serum  of  a  rabbit  that  has  been 
injected  with  normal  hen  blood.  Normal  hen  serum  has  the 
property  of  agglutinating  and  dissolving  rabbit  corpuscles.  It  may 
well  be,  then,  that  the  rabbit  that  has  been  " vaccinated"  against 
hen  blood  should  furnish  a  serum  that  is  able,  not  only  to  attack 
hen  red  blood  corpuscles  energetically,  but  also  to  defend  rabbit 
corpuscles  against  the  harmful  effect  of  hen  serum.  In  other  words, 
our  active  serum  might  be  endowed  to  a  certain  extent  with  anti- 
toxic properties  in  addition  to  the  antihematic  property,  which 
is  comparable  to  the  antibactericidal  property  of  cholera  serum; 
in  such  a  case  the  toxin  would  be  hen  serum.  We  shall  consider 
these  sera,  then,  from  two  standpoints,  first  as  regards  their  anti- 
hematic  property  and  secondly  as  regards  their  antitoxic  property. 

A.  Antihematic  property.  —  The  antihematic  property  is  present 
in  the  serum  of  animals  treated  with  several  injections  of  defibrinated 
blood  from  a  different  animal  species.*  The  serum  of  rabbits  that 
have  received  intraperitoneally  several  injections  of  10  c.c.  each  of 
defibrinated  hen  blood  shows  properties  similar  to  those  found  in 
the  serum  of  guinea-pigs  treated  with  rabbit  blood.  Although 
normal  rabbit  serum  has  only  the  faintest  agglutinating  and  dis- 
solving effect  on  hen  red  blood  corpuscles  this  active  serum  agglu- 
tinates and  dissolves  them  energetically.  This  action,  however,  is 

*  Rabbits  that  have  received  six  intraperitoneal  injections  of  10  c.c.  of  defibri- 
nated rabbit  blood  show  no  particular  property  in  their  serum.  This  is  reasonable 
enough.  The  production  of  the  active  substances  found  in  serum  is  evidently  due 
to  a  stimulation  of  the  cells  in  the  animal  body  on  the  introduction  of  foreign 
substances  not  normally  present,  which  may  have  some  effect  on  the  cell,  or, 
in  other  words,  which  may  lead  to  a  change  in  the  chemical  or  physical  constitution 
of  the  cell. 


AGGLUTINATION   AND   DISSOLUTION.  167 

only  on  the  protoplasm  of  the  corpuscle  and  does  not  affect  the 
nucleus. 

A  microscopical  examination  of  a  mixture  of  hen  blood  and 
active  serum  shows  that  the  red  blood  corpuscles  are  clumped  in 
more  or  less  compact  masses  and  reduced  to  their  nuclei.  Nothing 
remains  of  the  protoplasm  but  a  very  delicate  stroma.  On  stain- 
ing with  eosin  followed  by  methylene  blue  no  protoplasmic  out- 
line is  visible;  the  affinity  of  protoplasm  for  eosin  has  disappeared; 
the  nuclei,  however,  stain  blue  as  usual.  Such  lesions  of  the 
red  blood  corpuscles,  however,  are  not  characteristic  of  an  active 
specific  serum  alone,  but  may  occur  when  hen  corpuscles  are 
placed  in  contact  with  a  sufficiently  active  normal  serum,  as  for 
example  dog  serum.  The  active  serum  is  distinguished  only  by 
the  remarkable  intensity  of  its  properties  against  the  corpuscles  in 
question. 

The  destructive  property  is  destroyed  on  heating  the  serum  to 
55  degrees,  but  may  be  restored  by  the  addition  of  fresh  normal 
rabbit  or  guinea-pig  serum  to  the  heated  serum.  This  fresh  normal 
serum  contains,  as  we  know,  alexin.*  The  heated  active  serum  re- 
tains both  its  agglutinating  property  and  the  property  of  forming 
with  alexin  a  mixture  that  has  intense  hemolytic  power.  We  shall 
not  insist  further  on  the  significance  of  these  facts  nor  on  the  analo- 
gies between  antimicrobial  sera  and  antihematic  sera  in  this 
respect,  since  they  have  already  been  pointed  out  in  our  preceding 
article. 

If  defibrinated  hen  blood,  to  which  heated  active  serum  has  been 
added,  is  introduced  into  the  peritoneal  cavity  of  a  normal  rabbit, 
a  rapid  destruction  of  the  protoplasm  of  the  corpuscles  may  be 
noted  under  the  influence  of  the  alexin  from  the  peritoneal  exudate. 
The  nuclei  of  the  corpuscles,  deprived  of  their  protoplasm  and 
resisting  the  action  of  the  body  fluid  in  vivo  as  well  as  in  vitro,  are 
found  in  such  an  exudate.  These  nuclei  are  later  taken  up  by  the 
macrophages  of  the  peritoneal  cavity. 

The  question  arises  as  to  whether  an  antihematic  serum  from  a 
treated  animal  will  endow  a  normal  animal  of  the  same  species  with 
a  " passive  immunity"  analogous  in  its  characteristics  to  that 

*  The  normal  serum  may  be  replaced  by  the  peritoneal  exudate  from  a  normal 
rabbit  which  also  contains  alexin. 


168  STUDIES  IN   IMMUNITY. 

afforded  by  injecting  antibacterial  sera.  We  know  that  cholera 
serum  when  injected  into  a  normal  animal  causes  a  remarkable 
phenomenon :  the  serum  of  the  treated  animal  becomes  bactericidal 
for  the  cholera  vibrio.*  It  is  important  to  determine  whether  the 
serum  from  a  rabbit  injected  with  a  serum  active  against  hen  cor- 
puscles becomes  destructive  for  these  corpuscles. 

A  small  amount  of  blood  is  taken  from  a  normal  rabbit  and  gives 
serum  A.  After  bleeding,  the  rabbit  is  given  10  c.c.  of  active  serum 
subcutaneously.  On  the  following  day  the  animal  is  bled  again 
and  serum  B  is  obtained  from  this  bleeding.  The  hemolytic  proper- 
ties of  serum  A  may  be  compared  with  those  of  serum  B.  It  is 
found  that  serum  B  has  a  distinct  hemolytic  property  for  hen  cor- 
puscles, whereas  serum  A,  from  the  normal  rabbit,  has  only  the 
faintest  activity.  Serum  B  also  has  an  agglutinating  property, 
which,  however,  is  slight.  This  property  has  been  transmitted, 
although  much  weakened  by  dilution  in  the  fluids  of  the  normal 
rabbit.  It  is  found,  indeed,  that  although  the  active  serum  em- 
ployed agglutinates  three  or  four  volumes  of  hen  blood  if  diluted 
with  20  to  25  parts  of  normal  salt  solution  it  causes  no  rapid  agglu- 
tination in  any  dose.f  We  have  already  noted  the  fact  that  agglu- 
tinins  when  injected  subcutaneously  into  a  normal  animal  go  into 
the  blood  rapidly.J 

The  most  natural  conclusion  to  be  drawn  from  this  experiment 
is  that  the  active  substances  in  serum,  when  injected  subcutaneously, 
are  simply  diluted  in  the  fluids  of  the  recipient.  It  follows,  there- 
fore, that  the  serum  of  this  latter  animal  should  acquire  to  a  less 
degree  all  the  properties  which  characterize  the  active  serum  in- 
jected. And  this,  in  fact,  is  what  happens.  When  heated  to  55 
degrees,  serum  B  loses  its  destructive  property,  but  recovers  it  on  the 
addition  of  a  little  of  serum  A  (normal  serum).  It  is  scarcely 
necessary  to  note  that  experiments  such  as  these  are  controlled 

*  This  important  fact  was  noted  for  the  first  time  by  Fraenkel  and  Sobernheim, 
Hygienische  Rundschau,  1894,  No.  1. 

t  Under  these  conditions  the  agglutination  occurs  only  after  a  considerable 
period.  The  same  is  true  of  serum  B.  When  agglutination  is  very  slow  heated 
sera  must  be  used. 

I  See  article,  page  56.  Agglutinins  injected  under  the  skin  are  also  found  in 
the  peritoneal  exudate,  but  in  smaller  amounts  than  in  the  blood.  (The  experi- 
ment to  demonstrate  this  fact  was  done  with  cholera  serum.) 


AGGLUTINATION  AND  DISSOLUTION.  169 

with  accuracy  as  to  dosage.     For  example,  in  such  an  experiment 
the  following  mixtures  would  be  made: 

Mixture  (a) :  one  part  of  defibrinated  hen  blood;  four  parts  of  serum  A  (heated 
for  one-half  hour  to  55  degrees) ;  three  parts  of  unheated  serum  A.  This  is  the 
control. 

Mixture  (6):  one  part  of  defibrinated  hen  blood;  four  parts  of  serum  B  (55 
degrees) ;  three  parts  of  unheated  serum  A. 

A  drop  from  each  mixture  is  suspended  on  a  hollow  ground  slide.  In  mixture 
(a)  the  corpuscles  remain  intact.  On  the  following  day  a  few  free  nuclei  are  found, 
but  the  destroyed  corpuscles  are  in  the  great  minority.  As  we  already  know, 
normal  rabbit  serum  has  a  slight  hemolytic  activity.  In  mixture  (6)  the  destruc- 
tion of  red  blood  cells  occurs  rapidly,  so  that  at  the  end  of  an  hour  there  are  nothing 
but  free  nuclei  left. 

In  comparing  the  dissolving  activity  of  different  fluids  in  such 
an  experiment,  care  must  be  taken  to  keep  them  at  the  same  tem- 
perature. The  dissolution  of  red  blood  cells  is  very  much  accel- 
erated by  heat,  as  is  the  granular  transformation  of  the  cholera 
vibrio. 

As  this  experiment  clearly  shows,  there  is  no  reason  to  suppose 
that  the  injection  of  active  serum  into  normal  animals  brings  about 
any  secretion  of  a  particular  dissolving  alexin  which  is  not  present 
in  normal  serum.  To  all  intents  the  substances  injected  have  been 
simply  diluted  in  the  body  fluids  of  the  recipient.  Any  experiments 
that  can  be  performed  with  active  serum  may  also  be  done  with  the 
serum  of  a  passively  immunized  animal. 

Consequently,  if  we  regard  the  proper  substances  of  the  active 
serum  as  simply  diluted  in  the  body  fluids  without  any  further 
change  on  injection  into  normal  animals,  we  should  also  expect  to 
obtain  a  fluid  which  is  exactly  as  active  as  serum  B,  by  mixing  serum 
A  with  a  certain  amount  of  the  active  serum  employed.  Such  a 
mixture  of  active  serum  and  serum  A,  should  be  made,  to  be  sure, 
in  proportions  that  are  comparable  to  the  relation  between  the 
amount  of  active  serum  injected  and  the  amount  of  fluid  in  the 
animal  body. 

This  is  found  to  be  true.  In  the  preceding  experiment  serum 
B  (which  was  obtained  from  a  rabbit  that  weighed  about  2000 
grams  and  had  received  10  c.c.  of  active  serum)  shows  activity 
similar  to  that  obtained  by  diluting  1  part  of  active  serum  with 
20  parts  of  serum  A. 


170  STUDIES  IN   IMMUNITY. 

This  experiment  may  be  done  more  exactly  as  follows:  A  normal  guinea-pig 
of  600  grams  is  given  3  c.c.  of  guinea-pig  serum  active  against  rabbit  corpuscles. 
A  normal  serum  has  been  previously  obtained  from  this  guinea-pig  before  injec- 
tion (serum  A).  Serum  B  is  obtained  by  bleeding  the  guinea-pig  24  hours  after 
injection.  A  mixture  is  made  of  one  part  of  active  serum  (the  same  as  used  for 
injection)  and  nineteen  parts  of  serum  A  (equals  serum  C).  This  mixture  and 
also  serum  B  are  then  heated  for  half  an  hour  to  55  degrees  to  destroy  the  alexin, 
since  the  amount  of  alexin  in  these  two  fluids  might  differ.  A  comparison  of  the 
power  of  sera  B  and  C  to  form  a  dissolving  mixture  for  rabbit  corpuscles  with 
normal  serum  A  containing  alexin,  may  then  be  made.  This  is  done  by  deter- 
mining the  smallest  amount  of  B  and  of  "C  respectively,  which,  in  the  presence 
of  a  given  amount  of  serum  A,  will  completely  dissolve  a  given  quantity  of  red 
blood  cells.  B  is  found  to  be  slightly  less  active  than  C.  From  the  result  we  should 
conclude  that  the  active  serum  when  injected  subcutaneously  in  a  guinea-pig  of 
600  grams  is  simply  diluted  in  slightly  more  than  60  c.c.  of  body  fluid.  This 
dilution  corresponds  pretty  well  to  what  actually  takes  place. 

The  different  facts  considered  up  to  this  point,  namely,  concerning 
the  identity  in  action  of  the  sera  on  corpuscles  whether  in  vivo  or 
in  vitro,  lead  us  to  the  conclusion  previously  offered  for  antimicro- 
bial sera.  Antihematic  sera  have  no  particular  cellulicidal  sub- 
stance; the  cellulicidal  substance  (alexin)  destroyed  at  55  degrees 
is  present,  not  only  in  immunized  animals,  but  also  in  normal  animals. 
Although  it  is  only  slightly  active  in  normal  serum  it  acts  energeti- 
cally in  the  serum  of  treated  animals  because  it  occurs  there  in  con- 
junction with  the  proper  substance  of  this  active  serum  which 
resists  heating  to  55  degrees  or  60  degrees  and  favors  the  action  of 
the  alexin.  The  intense  cellulicidal  power  which  appears  in  the 
serum  of  a  normal  animal  following  the  injection  of  active  serum 
is  not  due  to  a  reaction  on  the  part  of  the  animal  nor  to  any  trans- 
formation of  the  substance  injected,  but  simply  to  the  encounter 
within  the  animal  body  of  the  two  substances  that  are  necessary  to  form 
intense  cellulicidal  power.  The  animal  already  contained  one  of 
-the  substances,  the  alexin,  and  the  other,  a  specific  substance 
characteristic  of  active  serum,  is  furnished  by  the  injection.  The 
union  of  these  two  substances,  which  occurs  in  the  animal  body, 
may  also  be  produced  in  vitro  by  simply  mixing  normal  serum  with 
either  intact  or  previously  heated  active  serum.* 

*  We  shall  not  consider  at  this  point  in  all  its  details  the  conclusions  that 
we  offered  in  1895  concerning  antimicrobial  sera  (see  p.  79)  applicable  in  this 
instance  to  antihematic  sera. 


AGGLUTINATION  AND  DISSOLUTION.  171 

The  specificity  of  rabbit  serum  active  against  hen  blood. — We  have 
not  done  many  experiments  on  this  subject,  but  it  seems  certain 
that  this  serum  is  quite  as  specific  from  all  appearances  as  are 
antimicrobial  sera.  This  active  rabbit  serum  naturally  enough  has 
no  effect  on  rabbit  corpuscles  and  it  also  has  no  more  effect  on 
guinea-pig  corpuscles  than  has  normal  rabbit  serum;  nor  does  it 
have  a  distinctive  action  on  pigeon,  human  or  mouse  corpuscles. 
With  none  of  these  corpuscles  is  the  agglutination  and  energetic 
dissolution  that  occurs  with  hen  corpuscles  to  be  noted. 

The  fixation  by  corpuscles  of  the  specific  substances  of  antihematic 
serum.  —  Corpuscles  that  are  susceptible  to  a  given  antihematic 
serum  fix  the  active  substances  of  this  serum.  We  have  already 
considered  this  fact  in  a  recent  article;*  and  have  also  noted  that 
bacteria  absorb  all  the  active  substances  from  antimicrobial  sera  in  a 
similar  manner.  Corpuscles  that  are  not  affected  by  a  serum  do  not 
absorb  its  active  properties  and  this  remark  applies  to  normal  sera 
as  well  as  to  specific  sera.  For  example,  if  normal  hen  serum  is 
added  to  rabbit  corpuscles  the  corpuscles  are  energetically  agglu- 
tinated ;  and  the  supernatant  fluid  is  found  to  be  deprived  of  agglu- 
tinating properties  for  fresh  rabbit  corpuscles.  But  if  hen  serum 
is  placed  in  contact  with  corpuscles  that  it  affects  only  slightly,  for 
example,  with  guinea-pig  corpuscles,  the  supernatant  fluid  still 
retains  its  agglutinating  property  for  susceptible  corpuscles. 

The  technique  employed  in  absorbing  the  active  principles  of  an 
antihematic  serum  follows :  the  active  serum  employed  is  heated  to 
55  degrees  for  half  an  hour  and  thereby  deprived  of  its  dissolv- 
ing effect.  The  following  mixtures  are  then  made  in  test  tubes: 

A,  3  c.c.  of  normal  rabbit  serum  plus  0.5  of  a  cubic  centimeter  of  active  guinea- 
pig  serum. 

B,  3  c.c.  of  defibrinated  rabbit  blood  plus  0.5  of  a  cubic  centimeter  of  active 
guinea-pig  serum. 

The  first  tube  is  a  control.  Both  tubes  contain  equal  amounts  of  active  serum 
diluted  in  a  similar  manner.  The  second  tube,  however,  contains  corpuscles, 
whereas  the  first  does  not.  After  contact  the  tubes  are  centrifugalized  and  the 
supernatant  fluids  decanted.  The  clear  fluids  thus  obtained  are  tested  for  agglu- 
tinating property.  Fluid  A,  which  has  not  been  subjected  to  corpuscles,  aggluti- 
nates rabbit  corpuscles  energetically  whereas  fluid  B  does  not  agglutinate  them. 
A  determination  is  then  made  of  the  power  of  the  two  fluids  to  form  hemolyzing 
mixtures  with  alexin,  as  follows: 

*  Mechanism  of  agglutination,  p.  142. 


172  STUDIES  IN   IMMUNITY. 

a,  one  part  of  normal  defibrinated  rabbit  blood  plus  one  part  of  fluid  A  plus 
five  parts  of  fresh  normal  guinea-pig  serum. 

6,  one  part  of  normal  defibrinated  rabbit  blood,  one  part  of  fluid  B,  five  parts 
of  fresh  normal  guinea-pig  serum. 

Dissolution  occurs  rapidly  in  tube  a,  but  the  corpuscles  remain  intact  in 
tube  b. 

It  is  evident  that  red  blood  corpuscles  fix  the  active  substances 
in  antihematic  sera  with  avidity.  It  is  also  found  that  the  effect 
of  these  substances  on  the  corpuscles  that  leads  to  their  agglutina- 
tion and  sensitization  to  alexin  is  very  profound,  since  it  is  not 
eliminated  by  several  washings.  These  washings  are  easily  accom- 
plished. Several  drops  of  salt  solution  containing  numerous  cor- 
puscles are  placed  in  a  test  tube*  and  a  small  amount  of  active 
serum,  heated  to  55  degrees  and  so  deprived  of  dissolving  effect, 
is  added.  The  tube  is  filled  .with  salt  solution,  shaken  and  cen- 
trifugalized ;  the  supernatant  fluid  is  decanted;  fresh  salt  solution 
is  added  to  the  deposition  of  corpuscles  and  the  washing  repeated 
several  times  until  a  deposit  of  well-washed  corpuscles  is  obtained. 

These  washed  sensitized  corpuscles  are  as  easily  dissolved  by  a 
dose  of  fresh  normal  serum  as  are  unwashed  sensitized  corpuscles. 

These  facts  seem  to  prove  that  the  particular  substance  present 
in  the  serum  of  vaccinated  animals  which  resists  heat  and  permits 
the  energetic  action  of  the  alexin  acts  upon  the  corpuscles  themselves 
in  such  a  way  as  to  sensitize  them  to  the  action  of  the  alexin.  We 
might  have  supposed,  indeed,  that  this  particular  substance  acted, 
not  on  the  corpuscles,  but  directly  on  the  alexin  in  a  way  to  render 
it  more  energetic.  This  latter  hypothesis,  however,  is  not  in  accord- 
ance with  facts  and,  moreover,  would  not  explain  so  well  the  evident 
specificity.  It  would  seem  to  be  demonstrated  by  the  preceding 
experiment  that  in  a  mixture  of  corpuscles,  active  heated  serum,  and 
normal  serum  there  is  no  reaction  in  the  fluid  part  of  the  mixture 
between  the  alexin  and  the  particular  substance  of  the  active  serum. 
This  is  shown  by  the  fact  that  the  specific  substance  rapidly  sepa- 
rates from  the  fluid  and  fixes  itself  on  the  corpuscles,  which  then 
become  susceptible  to  the  subsequently  added  alexin;  this  is  shown 
by  adding  it  only  after  the  sensitizing  substance  has  been  combined 

*  In  such  an  experiment  the  corpuscles  employed  have  been  previously  washed 
to  remove  the  serum  present  in  defibrinated  blood;  in  such  an  experiment  no  trace 
of  alexin  should  be  left. 


AGGLUTINATION   AND  DISSOLUTION.  173 

with  the  corpuscles.  Similar  experiments  may  be  made  with  bac- 
teria and  their  corresponding  antisera.*  We  may  consider,  then,  the 
conception  that  we  previously  arrived  at,  namely,  that  specific  sera 
contain  a  sensitizing  substance  that  renders  the  corpuscles  or  bacteria 
susceptible  to  attack  on  the  part  of  the  alexin,  as  sufficiently  proved. 

We  have  not  yet  considered  the  close  analogies  between  the 
properties  in  the  serum  of  a  guinea-pig  active  against  rabbit  blood 
and  those  present  in  the  serum  of  a  rabbit  active  against  hen  blood. 
There  is,  however,  a  difference  between  these  two  sera  that  should 
be  noted. 

As  we  have  already  noted  in  a  previous  article,  active  guinea-pig 
serum  heated  to  55  degrees  when  added  to  defibrinated  rabbit  blood 
still  produces  a  slight  effect  on  these  corpuscles,  as  is  shown  by  a 
distinct  reddish  coloration  of  the  fluid.  This  is  due,  as  we  have 
already  noted,  to  the  fact  that  these  corpuscles  are  slightly  attacked 
by  the  small  amount  of  rabbit  alexin  present  in  the  defibrinated 
blood.  If,  indeed,  corpuscles  previously  washed  in  salt  solution  are 
used  instead  of  ordinary  defibrinated  blood,  no  dissolution  takes 
place;  that  is  to  say,  the  alexin  has  been  eliminated  by  washing. 
On  the  other  hand,  if  to  such  washed  corpuscles,  subsequently  sen- 
sitized, a  sufficient  dose  of  normal  rabbit  serum  is  added,  complete 
and  rapid  dissolution  occurs.  Rabbit  corpuscles,  then,  under  the 
influence  of  the  sensitizing  substance,  become  susceptible  to  the 
action  of  their  proper  alexin.  We  should  expect  that  hen  cor- 
puscles when  treated  with  active  heated  serum  (from  a  rabbit 
injected  with  hen  blood)  would  dissolve  in  normal  hen  serum.  Such 
dissolution,  however,  does  not- occur.  In  spite  of  the  effect  of  the 
sensitizing  substance  hen  corpuscles  do  not  become  susceptible  to 
hen  alexin. 

This  shows  clearly  enough  that  the  alexins  are  not  quite  identical 
in  different  animal  species.  Their  more  important  characteristics" 
are  common ;  that  is,  they  act  more  or  less  in  the  same  way  on  a 
given  bacterium,  but  they  show  differences  in  their  action  upon 
red  blood  cells.  The  idea,  moreover,  was  a  priori  evident  from  a 
study  of  the  effect  of  normal  sera  on  corpuscles,  from  which  we 

*  Cholera  vibrios  treated  with  cholera  serum  and  then  carefully  washed  show 
granular  transformation  upon  the  addition  of  normal  serum  or  when  introduced 
in  the  peritoneal  cavity  of  a  normal  guinea-pig. 


174  STUDIES  IN   IMMUNITY. 

learned  that  the  red  blood  cells  of  an  animal  are  not  attacked  by  the 
alexin  from  the  same  animal,  whereas  they  are  susceptible  to  alexins 
from  other  animals. 

We  might  compare  in  a  very  gross  way  the  modification  which 
the  sensitizing  substance  causes  in  the  corpuscles  by  likening  it 
to  a  change  in  the  structure  of  a  lock  by  means  of  which  one  or 
several  keys  that  previously  could  not  open  it  are  enabled  to  do  so. 
Any  two  keys  that  are  sufficiently  alike  would  enter  the  lock  in- 
differently. In  the  same  way  the  alexins,  both  of  the  guinea-pig 
and  of  the  rabbit,  "enter"  rabbit  corpuscles  or  hen  corpuscles 
once  these  corpuscles  have  been  sensitized.  On  the  other  hand, 
hen  alexin,  that  apparently  differs  too  greatly  from  guinea-pig  or 
rabbit  alexin,  does  not  " enter"  hen  corpuscles  even  when  they  are 
previously  sensitized.  It  may  be  that  the  entrance  or  the  efficient 
working  of  the  alexin  depends,  not  only  on  the  nature  of  the  alexin, 
but  also  on  the  nature  or  the  particular  origin  of  the  sensitizing  sub- 
stance. There  are  evidently  many  experiments  yet  to  be  made. 
In  the  absence  of  sufficient  data  we  shall  content  ourselves  for  the 
moment  with  pointing  out  a  method  of  obtaining  some  insight  into 
the  mode  of  action  of  these  substances. 

The  effect  of  heat  on  the  active  substances  of  serum.  —  On  heating 
rabbit  serum,  active  against  hen  corpuscles,  to  55  degrees  the  agglu- 
tinating and  sensitizing  properties  persist,  whereas,  as  we  have 
already  seen,  the  dissolving  property  is  suppressed. 

On  heating  for  half  an  hour  to  65  degrees,  both  the  agglutinating 
and  the  sensitizing  power  are  still  intact;  heating  to  65  degrees 
diminishes  the  agglutinating  property  slightly,  and  it  becomes  very 
weak  on  heating  to  70  degrees  for  the  same  length  of  time.  It  should 
be  remarked  at  this  point  that  unheated  serum  when  previously 
diluted  with,  say,  20  parts  of  salt  solution  has  no  visible  agglutina- 
ting effect  in  any  dose,  which  shows  that  the  agglutinin  must  be 
present  in  a  certain  state  of  concentration  in  order  to  affect  cor- 
puscles, and,  moreover,  when  the  phenomenon  is  absent  no  conclusion 
can  be  drawn  as  to  complete  absence  of  the  active  substance.  The 
sensitizing  substance,  however,  is  still  distinctly  manifest  in  serum 
heated  to  70  degrees  and  also  in  serum  that  has  been  diluted  with 
20  parts  of  salt  solution.  One  part  of  defibrinated  blood  mixed 
with  two  parts  of  serum  heated  to  70  degrees  plus  four  parts  of 


AGGLUTINATION  AND   DISSOLUTION.  175 

normal  rabbit  serum,  is  rapidly  hemolyzed.  But,  with  the  same 
amount  of  defibrinated  blood  and  normal  serum,  the  addition  of 
relatively  small  doses  of  active  serum  heated  either  to  60  degrees  or 
to  70  degrees  shows  that  dissolution  takes  place  more  slowly  with 
the  serum  heated  to  70  degrees;  that  is,  the  sensitizing  property  of 
the  serum  has  been  diminished  by  heating  to  70  degrees.  It  is  very 
attenuated,  but  is  not  completely  destroyed  by  heating  for  half 
an  hour  to  75  degrees. 

It  may  be  noted  that  a  mixture  of  defibrinated  hen  blood,  normal 
serum,  and  active  serum  heated  to  70  degrees  is  hemolyzed  with- 
out any  preliminary  agglutination;  the  corpuscles  are  soon  reduced 
to  separate  nuclei.  If  the  agglutinin  is  not  destroyed  it  has  at 
least  become  too  feeble  or  too  small  in  amount  to  produce  clump- 
ing; sensitization,  however,  still  goes  on. 

Precipitating  property.  —  In  addition  to  the  antihematic  property 
in  active  rabbit  serum  we  should  note  its  property  of  forming  a 
precipitate  with  normal  hen  serum.  We  have  already  considered 
this  question  in  a  preceding  article*  and  in  this  connection  we 
have  mentioned  the  researches  of  Tchistovitch,  to  whom  we  owe 
the  first  observations  of  a  similar  phenomenon.  When  the  two 
sera  are  mixed  a  turbidity  appears  that  at  first  is  slight  but  soon 
increases  and  finally  condenses  into  flecks.  The  most  abundant 
precipitate  is  formed  by  a  mixture  of  one  or  two  parts  of  hen  serum 
with  eight  or  nine  parts  of  active  serum. 

If,  instead  of  treating  rabbits  with  hen  blood,  hens  are  treated 
with  rabbit  blood,  their  serum  is  found  to  precipitate  normal  rabbit 
serum.  The  phenomenon  of  precipitation,  however,  does  not  occur 
regularly  in  all  instances  examined ;  the  serum  of  guinea-pigs  treated 
with  rabbit  blood,  for  example,  causes  no  precipitate  with  rabbit 
serum.  A  mixture  of  sera  from  normal  animals  of  different  species 
never  causes  a  precipitate. 

B.  Antitoxic  property.  —  It  seemed  probable  that  we  should  be 
able  to  produce  sera  endowed  with  an  antitoxic  property  and 
capable  of  opposing  the  destructive  action  of  certain  given  sera  on 

*  See  p.  142.  We  shall  not  here  consider  the  significance  of  this  phenomenon. 
We  have  already  mentioned  the  effect  of  heat  on  the  precipitating  property  and 
determined  to  what  extent  the  precipitins  are  specific.  We  may  recall  that  the 
serum  in  question  (serum  rabbit  >  hen)  also  precipitates  pigeon  serum. 


176  STUDIES  IN  IMMUNITY. 

red  blood  cells.  As  a  matter  of  fact,  Camus  and  Gley  and  Kossel 
have  shown  that  in  the  serum  of  animals  immunized  against  eel 
serum  there  are  substances  that  protect  their  corpuscles  against  the 
dissolving  effect  of  this  toxic  serum. 

In  order  to  make  such  results  evident  the  " toxin"  employed 
should  be  powerful.  Animals  of  species  A  should  be  injected  with 
serum  or  defibrinated  blood  of  species  B,  and  the  serum  of  species  B 
should  be  able  to  agglutinate  and  dissolve  the  red  blood  corpuscles 
of  species  A  energetically.  The  rabbit  and  the  hen  fulfill  these 
conditions:  the  agglutinating  and  dissolving  effect  of  normal  hen 
serum  on  rabbit  corpuscles  being  very  energetic.  The  antihematic 
property  of  rabbit  serum  for  guinea-pig  corpuscles,  on  the  contrary, 
is  only  slight;  and  therefore  the  serum  of  a  guinea-pig  vaccinated 
against  rabbit  blood  is  not  suited  to  the  study  of  this  antitoxic  or 
antihemotoxic  property. 

Normal  hen  serum  agglutinates  an  equal  amount  of  defibrinated 
rabbit  blood  energetically.  Two  or  three  parts  of  this  serum  will 
dissolve  one  part  of  rabbit  blood  corpuscles.  Rabbits  that  have 
received  intraperitoneally  three  injections  of  10  c.c.  each  of  hen 
blood  have  an  antitoxic  property  in  their  serum,  although  it  is  not 
very  intense.  It  may  be  demonstrated  in  a  mixture  containing 
normal  rabbit  blood,  hen  serum  (fresh)  and  active  rabbit  serum; 
and,  as  a  control,  normal  rabbit  blood,  fresh  hen  serum  and  normal 
rabbit  serum.  The  exact  proportions  in  these  tubes  are  as  follows: 

Tube  A,  one  part  of  normal  rabbit  blood;  three  parts  of  hen 
serum ;  ten  parts  of  active  rabbit  serum. 

Tube  B,  one  part  of  normal  rabbit  blood;  three  parts  of  hen 
serum;  ten  parts  of  normal  rabbit  serum.  In  the  second  of  these 
tubes  the  rabbit  blood  corpuscles  rapidly  collect  into  thick  compact 
masses  that  after  two  or  three  hours  hemolyze.  In  tube  "A"  the 
corpuscles  remain  indefinitely  intact  and  only  a  slight  agglutination 
in  very  small  clumps  is  evident.  In  addition  to  a  property  of  pre- 
venting the  dissolution  of  corpuscles  the  active  serum  has  a  distinct 
anti-agglutinating  property. 

The  control  mixture  "B"  shows  that  normal  rabbit  serum  has  no 
antitoxic  properties. 

If  the  amount  of  active  serum  is  diminished  to  any  extent  or  the 
amount  of  dissolving  hen  serum  is  increased,  the  antitoxic  effect 


AGGLUTINATION  AND  DISSOLUTION.  177 

is  no  longer  detectable.  So,  for  example,  in  a  mixture  of  one 
part  of  rabbit  blood,  three  parts  of  active  serum  and  six  parts  of 
hen  serum  the  corpuscles  are  dissolved  without  any  appreciable 
delay. 

In  order  to  determine  more  clearly  the  effect  of  the  "anti-agglu- 
tinin"  in  the  serum,  hen  serum  heated  to  55  degrees  and  therefore 
without  dissolving  property  may  be  used;  such  a  serum  still  agglu- 
tinates very  energetically  an  equal  volume  of  rabbit  blood.  For 
example,  the  following  mixtures  are  prepared : 

Tube  A,  defibrinated  blood,  one  part;  active  serum,  ten  parts; 
hen  serum,  55  degrees,  one  part. 

Tube  B,  defibrinated  blood,  one  part;  normal  rabbit  serum,  ten 
parts;  hen  serum,  55  degrees,  one  part. 

Agglutination  is  rapid  and  very  strong  in  mixture  "B"  and  is 
negative  or  extremely  feeble  in  mixture  "A." 

When  the  active  serum  is  mixed  with  hen  serum  a  precipitate 
is  formed.  We  might  imagine  that  the  active  serum  does  not  con- 
tain, strictly  speaking,  an  antitoxic  substance,  but  that  the  precip- 
itate in  its  formation  removes  the  harmful  substances  of  the  hen 
serum  and  so  renders  it  inactive.  This  objection,  however,  is  not 
well  founded,  as  is  shown  by  the  fact  that  active  rabbit  serum 
heated  to  70  degrees  loses  the  property  of  producing  a  precipitate 
with  serum,  but  still  manifests  its  anti-agglutinating  properties. 
It  retains,  moreover,  the  property  of  preventing  the  dissolution 
of  corpuscles  by  hen  alexin. 

This  shows  that  the  substance  that  opposes  destruction  by  alexin 
is  quite  different  from  alexins  themselves. 

The  serum  of  a  guinea-pig  vaccinated  against  rabbit  blood  also 
shows  antitoxic  properties,  but  only  to  a  slight  extent. 

II.  ANALOGIES  BETWEEN  SPECIFIC  AND  NORMAL  SERA.  ANAL- 
OGIES BETWEEN  SUBSTANCES  ACTIVE  AGAINST  BACTERIA 
AND  THOSE  AFFECTING  BLOOD  CORPUSCLES. 

It  is  well  known  that  the  various  properties  in  specfic  sera  are 
also  present  to  a  slight  degree  in  normal  sera.  They  are  manifest 
by  an  agglutinating  action  or  dissolving  action  either  on  bacteria 
or  on  corpuscles.  It  is  quite  natural  to  believe  that  the  properties 
acquired  by  the  animal  body  as  a  result  of  immunization  are  only 


178  STUDIES  IN   IMMUNITY. 

a  perfectioning  of,  or  an  increase  in,  preexisting  properties,  and  it 
is  therefore  of  interest  to  ascertain  whether  the  active  substances 
in  the  sera  of  vaccinated  animals  have  characters  in  common  with 
similar  substances  in  normal  sera  and  whether,  indeed,  the  two  are 
identical.  For  example,  we  may  compare  the  antihematic  proper- 
ties which  are  so  strongly  marked  in  the  serum  of  animals  treated 
with  defibrinated  blood  with  similar  properties  present  in  normal 
sera. 

The  property  of  agglutinating  and  dissolving  alien  red  blood 
cells  is  found  very  commonly  in  normal  sera.  We  know  that  these 
properties  affect  the  corpuscles  from  different  animal  species  only. 
Following  is  a  recapitulation  of  the  agglutinating  power  of  certain 
normal  sera  on  certain  varieties  of  red  blood  cells.  In  these  experi- 
ments we  usually  mixed  one  part  of  defibrinated  blood  with  three 
or  four  parts  of  serum : 

Guinea-pig  serum  agglutinates  the  corpuscles  of  the  rabbit  and 
the  hen  slightly  and  rat  corpuscles  more  energetically. 

Rabbit  serum  has  slight  agglutinating  properties  for  the  red 
blood  corpuscles  of  the  guinea-pig,  man,  the  hen  and  the  rat. 

Hen  serum  has  an  energetic  agglutinating  property  for  the  cor- 
puscles of  the  dog  and  rat  and  especially  for  the  rabbit.  Its  agglu- 
tinating action  on  guinea-pig  corpuscles  is  slight.  It  agglutinates 
pigeon  corpuscles  rather  strongly. 

Pigeon  serum  has  a  very  weak  agglutinating  property  for  the 
corpuscles  of  the  hen,  the  rabbit,  man,  and  also  for  bacteria. 

Dog  serum  agglutinates  the  corpuscles  of  the  rabbit  and  the 
rat  strongly,  and  the  corpuscles  of  the  guinea-pig  and  the  hen 
faintly. 

Rat  serum  agglutinates  the  corpuscles  of  the  rabbit  and  the 
guinea-pig  slightly. 

Goat  serum  agglutinates  the  corpuscles  of  the  rabbit  and  the 
guinea-pig  distinctly. 

Horse  serum  agglutinates  the  corpuscles  of  the  guinea-pig,  the 
rabbit  and  the  hen  distinctly  and  the  corpuscles  of  the  rat  ener- 
getically. 

The  dissolving  properties  of  these  animal  sera  for  red  blood 
corpuscles  are  always  due  to  the  alexin,  or  thermolabile  sub- 
stance. It  is  a  general  rule,  applicable  to  all  sera,  that  the 


AGGLUTINATION   AND   DISSOLUTION.  179 

power  to  destroy  red  blood  corpuscles  disappears  on  heating  to 
55  degrees.* 

The  most  destructive  and  powerful  action  that  we  have  noted  in 
studying  sera  is  the  effect  of  hen  serum  on  rabbit  corpuscles.  One 
part  of  defibrinated  rabbit  blood  is  rapidly  dissolved  by  two  or 
three  parts  of  hen  serum.  The  destructive  effect  of  this  serum  for 
rat  corpuscles  is  also  very  distinct;  it  is  very  slight  for  guinea-pig 
corpuscles. 

Dog  serum  dissolves  hen  corpuscles  energetically,  although  it 
leaves  their  nuclei  intact;  it  also  attacks  rabbit  and  guinea-pig  red 
blood  corpuscles. 

Guinea-pig  serum  and  rabbit  serum  have  both  a  very  slight 
destructive  effect  on  hen  and  human  corpuscles.  Guinea-pig  serum 
has  only  a  very  slight  and  delayed  effect  on  rabbit  corpuscles.  Rab- 
bit serum,  however,  is  much  more  active  against  guinea-pig  cor- 
puscles, but  is  almost  entirely  without  effect  on  rat  corpuscles. 

It  should  be  noted  in  these  tests  that  the  agglutinating  and  the 
dissolving  property  differ  in  different  specimens  of  the  same  variety 
of  normal  serum;  that  is,  there  are  individual  differences  which  may 
be  rather  marked. 

No  general  rules  can  be  drawn  from  these  facts.  There  is  no 
means,  for  example,  of  classing  corpuscles  in  groups  according  to 
their  sensitivity  to  agglutinating  or  dissolving  action. 

The  corpuscles  of  a  given  species  may  be  very  easily  agglutinated 
by  one  normal  serum  and  resist  another.  In  the  same  way  it  can- 
not be  shown  that  a  serum  that  shows  itself  very  active  for  one 
species  of  corpuscles  will  be  equally  active  for  other  varieties  of 
corpuscles.  For  example,  hen  serum  affects  rabbit,  dog  and  rat  cor- 
puscles, but  has  almost  no  effect  on  guinea-pig  corpuscles.  Nor 
can  we  assert,  as  a  general  rule,  that  a  serum  that  is  capable  of  dis- 
solving a  given  species  of  corpuscles  will  necessarily  agglutinate  them 
energetically.  For  example,  although  hen  serum  dissolves  rabbit 
corpuscles  easily  and  also  agglutinates  them  strongly,  we  find,  on 
the  other  hand,  that  dog  serum  agglutinates  hen  corpuscles  only 
faintly,  although  it  destroys  them  actively.  It  is  evident,  then,  that 

*  There  is,  however,  an  apparent  exception.  Dog  red  blood  corpuscles  are  still 
dissolved  by  sera  heated  to  55  degrees.  These  corpuscles,  however,  are  also  dis- 
solved by  their  proper  serum.  It  is,  therefore,  not  a  question  of  alexic  activity, 
but  of  a  particular  fragility  on  the  part  of  dog  corpuscles. 


180  STUDIES  IN  IMMUNITY. 

there  are  two  distinct  factors  concerned  in  the  effect  of  sera  on  cor- 
puscles: First,  the  greater  or  lesser  amount  of  active  substances  in 
the  serum  and,  secondly,  the  particular  sensitivity  of  the  corpuscles 
in  question  to  the  substances  in  the  serum  employed.  Corpuscles 
may  be  very  susceptible  to  the  effect  of  the  active  substances  of  one 
serum  and  much  more  refractory  to  those  in  another  normal  serum. 
Although  the  agglutinins  in  different  normal  sera  are  similar  in  their 
method  of  action  they  are  not  identical;  there  must  be  certain  slight 
differences  of  chemical  constitution  between  them  to  explain  the 
diversity  of  their  effect  on  a  given  kind  of  corpuscles.  The  same  re- 
marks apply  also  to  the  alexins  from  different  animal  species.  The 
corpuscles,  too,  of  different  species  differ  in  constitution  and  each 
variety  has  its  particular  way  of  reacting  with  a  given  alexin  or  a 
given  agglutinin. 

We  have,  moreover,  offered  in  our  memoir  on  the  mechanism  of 
agglutination  an  experiment  which  indicates  that  a  given  normal 
serum  contains  several  different  agglutinins. 

*** 

It  is  well  to  consider  how  great  a  resemblance  there  is  between 
substances  with  similar  properties  in  different  sera.  For  example, 
we  may  compare  the  agglutinins  of  normal  sera  with  those  of  im- 
mune sera  and  the  agglutinins  and  sensitizing  substances  for  blood 
corpuscles  with  the  corresponding  substances  for  bacteria. 

We  have  already  frequently  repeated  that,  in  the  matter  of  alexins, 
normal  sera  do  not  differ  from  immune  sera,  whether  antimicrobial 
or  antihematic. 

And,  what  is  more,  the  alexins  that  affect  corpuscles  seem  to  be 
quite  identical  with  those  that  affect  bacteria.  Both  the  property 
of  changing  red  blood  corpuscles  and  the  power  of  producing  a  gran- 
ular transformation  of  vibrios,  are  frequently  met  with  among  sera. 
Vibrios,  especially  when  sensitized  by  cholera  serum,  are  readily 
changed  into  granules  when  placed  in  contact  with  normal  serum 
from  the  rabbit,  guinea-pig,  goat,  hen,  dog,  rat  or  pigeon;  only  a 
slight  difference  in  intensity  of  action  is  shown  between  these 
different  sera.*  These  sera  lose  their  activity  against  bacteria  when 

*  We  are  dealing  here,  it  may  be  stated,  with  those  bactericidal  effects  which 
depend  on  alexins  and  which  do  not  occur  with  sera  heated  to  55  degrees.  The 
typical  example  of  this  activity  is  the  granular  transformation  of  vibrios.  There 


AGGLUTINATION  AND   DISSOLUTION.  181 

heated  to  55  degrees;  and  at  the  same  time  are  deprived  of  their 
activity  against  corpuscles.  The  differences  in  alexins  from  dif- 
ferent sources,  so  far  as  their  action  on  corpuscles  is  concerned,  are 
not  to  be  reckoned  with  when  dealing  with  vibrios. 

Although  the  presence  of  alexins  is  not  distinctive  of  the  sera  of 
vaccinated  animals,  since  they  occur  in  the  same  form  in  normal 
serum,  the  agglutinating  properties  of  the  serum  of  vaccinated 
animals  are  clearly  to  be  distinguished  from  those  of  normal  sera  as 
more  intense  and  specific.  It  is  worth  considering,  however,  whether 
the  agglutinins  in  immunized  animals,  in  spite  of  these  differences, 
are  affected  in  the  same  way  by  heat  as  are  the  agglutinins  of 
normal  sera. 

We  may  begin  by  establishing  to  what  extent  heat  weakens  the 
agglutinating  power  of  a  given  normal  serum  for  corpuscles  and 
for  bacteria  respectively.  For  such  an  experiment  bacilli  easily 
clumped  by  various  normal  sera  are  chosen.  We  have  particularly 
employed  the  bacillus  typhosus.  Without  going  into  the  details 
of  these  experiments  we  may  say  in  general  that  a  given  high  tem- 
perature causes  a  normal  serum  to  lose  its  property  of  agglutinating 
both  bacteria  and  cells  to  an  equal  extent.  In  the  sera  that  we 
have  tried  the  agglutinating  properties  remain  unaffected  after 
heating  to  55  degrees  for  half  an  hour.  Heating  to  61  to  62  degrees 
for  the  same  period  diminishes  but  does  not  entirely  destroy  the 
agglutinating  properties  of  guinea-pig,  goat,  dog,  horse  and  hen 
serum.  With  poorly  agglutinating  sera  there  is  practically  no 
clumping  action  after  heating  to  this  degree;  when  dealing  with 
strongly  agglutinating  sera  it  is  still  very  distinct  after  such  treat- 
ment. Hen  serum,  for  example,  even  after  heating  to  67  degrees 
still  agglutinates  rabbit  corpuscles  distinctly.  As  the  temperature 
is  raised  the  agglutinating  power  becomes  weaker  and  weaker,  so 
that  normal  sera  heated  for  a  half  hour  to  70  degrees  no  longer 
agglutinate. 

are,  however,  in  certain  sera  bactericidal  substances  quite  distinct  from  alexins. 
Rat  serum  heated  to  55  degrees  is  quite  incapable  of  producing  a  transformation 
in  cholera  vibrios,  sensitized  with  cholera  serum,  although  this  property  is  present 
in  intact  rat  serum.  But  such  heated  rat  serum  destroys  the  anthrax  bacillus 
with  apparently  undiminished  activity.  Sawtchenko  has  already  noted  that  this 
particular  bactericidal  property  of  rat  serum  for  the  anthrax  bacillus  resists 
heating  to  from  55  to  60  degrees. 


182  STUDIES  IN   IMMUNITY. 

Normal  rabbit  serum  is  the  one  in  which  agglutinins  best  resist 
heating,  so  far  as  we  have  studied  them.  Rabbit  serum  must  be 
heated  to  approximately  65  degrees  to  cause  any  distinct  diminu- 
tion in  agglutinating  power  either  for  corpuscles  or  bacteria. 

When  we  come  to  compare  the  effect  of  heat  on  the  normal  and 
on  the  specific  agglutinins  we  find  very  distinct  correspondence. 
In  such  experiments  it  should  be  remembered  that  specific  agglu- 
tinins are  very  much  more  energetic  and,  consequently,  any  dimin- 
ution in  their  activity  is  less  evident  than  in  normal  sera,  the 
energy  of  which  is  slight  to  begin  with.  In  experiments  on  the 
sera  of  the  goat,  rabbit,  or  guinea-pig  vaccinated  against  the  cholera 
vibrio,  and  the  serum  of  the  dog  vaccinated  against  B.'  typhosus, 
the  specific  agglutinating  property  is  distinctly  diminished  by  any 
temperature  that  weakens  a  normal  agglutinating  power;  in  the 
case  of  the  guinea-pig  and  the  goat  this  diminution  is  evident  after 
heating  to  61  to  62  degrees;  and  the  sera  heated  to  70  degrees  are 
almost  inactive.  The  agglutinins  in  immune  rabbit  serum  are 
much  more  resistant;  and,  as  we  have  just  seen,  the  same  fact  holds 
true  for  the  normal  agglutinins  in  normal  rabbit  serum.  Two 
cholera  sera  coming  from  a  rabbit  and  a  guinea-pig  respectively 
resist  heat  differently;  on  heating  them  to  70  degrees  there  is  marked 
diminution  of  activity  in  the  specific  guinea-pig  serum,  but  only  a 
very  faint  diminution  in  the  specific  rabbit  serum.  It  may  be 
noted  that  rabbit  serum  also  resists  coagulation  by  heat  better  than 
does  guinea-pig  serum.  Guinea-pig  serum  mixed  with  equal  parts 
of  normal  salt  solution  becomes  opalescent,  but  is  not  coagulated, 
on  heating  to  65  degrees  for  a  half  hour;  rabbit  serum  treated  in 
the  same  way  does  not  even  become  opalescent;  it  becomes  faintly 
opalescent  only  on  heating  to  70  degrees.  We  need  scarcely  em- 
phasize the  fact  that  there  is  apparently  some  close  relation  between 
the  appearance  of  a  coagulation  or  opalescence  and  the  deteriora- 
tion of  active  substances. 

In  short,  it  may  be  said  that  agglutinins,  whether  affecting  cor- 
puscles or  bacteria,  are  similarly  affected  by  heat  whether  they  occur 
in  normal  sera  or  in  specific  sera.  There  is  a  progressive  diminution 
in  activity  as  the  temperature  is  raised,  but  it  is  impossible  to  define 
a  critical  temperature  below  which  they  remain  intact  and  above 
which  they  are  destroyed.  Agglutinins  appear  to  be  more  resistant 


AGGLUTINATION   AND   DISSOLUTION.  183 

in  the  serum  of  certain  animals  than  they  are  in  the  serum  of  other 
animal  species. 

Since  heat  weakens  the  agglutinating  property  of  sera  for  cor- 
puscles or  bacteria,  the  question  arises  as  to  whether  the  property 
of  sensitizing  cells  to  the  action  of  alexin  present  in  specific  sera 
is  diminished  in  a  similar  way.  The  question  is  not  without  in- 
terest, as  it  may  throw  some  light  on  the  problem  of  whether  the 
phenomenon  of  sensitization  is  due  to  the  same  or  different  sub- 
stances than  the  phenomenon  of  agglutination.  We  shall  not  under- 
take to  consider  this  subject  here,  but  reserve  for  a  future  time 
a  discussion  of  the  significance  of  facts  bearing  on  this  question.* 

We  shall  content  ourselves  with  noting  that  the  exposure  of  specific 
sera  to  temperatures  ranging  from  65  to  75  degrees,  which  distinctly 
enfeebles  their  agglutinating  and  immobilizing  poiuer,  also  distinctly 
diminishes  their  sensitizing  power.  When  cholera  serum  from  the 
rabbit  has  been  heated  to  70  degrees  it  is  less  energetically  agglu- 
tinating for  the  vibrio  and  is  also  slightly  less  sensitizing.  Such  a 
diminution  in  strength  may  be  demonstrated  by  placing  vibrios  in 
contact  with  small  doses  of  the  serum  heated  to  55  degrees  and  to 
70  degrees  respectively.  These  emulsions  are  not  equally  well  agglu- 
tinated. On  adding  normal  serum  to  each  emulsion  it  is  found 
that  the  granular  transformation  is  much  less  extensive  in  the 
emulsion  containing  serum  heated  to  70  degrees  than  in  the  one 
with  serum  heated  to  55  degrees.  A  similar  difference  in  trans- 
formation may  be  noted  in  vivo  on  intraperitoneal  injection  of  the 
emulsion.  Guinea-pigs  receiving  the  emulsion  with  serum  heated 
to  55  degrees  have  an  exudate  showing  a  granular  transformation 
of  the  vibrios,  and  the  animals  recover;  in  the  other  animals  motile 
vibrios  remain  and  a  fatal  infection  ensues.  If  more  than  minimal 
doses  of  serum  are  employed,  the  difference  between  the  two  emul- 
sions naturally  is  not  so  distinct ;  we  may  repeat  that  the  weakening 
effect  of  heat  on  the  properties  of  the  serum  is  only  partial.  A 

*  Agglutination  is  a  complex  phenomenon.  For  example,  when  motile  bacteria 
are  concerned  agglutination  includes  and  necessitates  an  immobilization  of  these 
bacteria.  It  has  not  been  demonstrated,  so  far,  whether  the  two  phenomena 
of  immobilization  and  agglutination  are  due  to  the  same  substance.  It  would 
seem  probable  when  we  consider  the  experiments  that  we  have  already  performed 
and  particularly  those  dealing  with  the  effect  of  salt  solution  on  agglutination  that 
agglutination  depends  on  the  cooperation  of  several  different  factors. 


184  STUDIES  IN   IMMUNITY. 

similar  diminution,  as  already  stated,  may  be  noted  on  heating 
rabbit  antihen  serum  to  a  temperature  of  70  degrees. 

The  sensitizing  property  of  cholera  serum  from  the  guinea-pig 
is  weakened  by  heating  to  a  lower  temperature  than  70  degrees,  as 
is  true  of  its  immobilizing  and  agglutinating  property ;  the  weaken- 
ing, indeed,  is  very  distinct  even  on  heating  to  65  degrees. 

Are  there  sensitizing  properties  in  normal  sera?  Is  not  the  action 
of  the  alexin  in  these  sera  aided  by  some  other  substances?  It  is 
difficult  to  answer  these  questions  because,  so  far,  we  have  not  suc- 
ceeded in  separating  alexins  from  the  other  substances  present  in 
serum.  On  combining  two  normal  sera,  however,  the  existence  of 
a  sensitizing  property  may  sometimes  be  shown.  For  example, 
cholera  vibrios,  immobilized  and  agglutinated  by  horse  serum,  are 
easily  transformed  to  granules  on  the  addition  of  normal  guinea- 
pig  serum.  No  generalized  statement,  however,  should  be  made  on 
the  basis  of  this  observation;  it  is  not  safe  to  conclude  that  it  suffices 
to  agglutinate  bacteria  or  corpuscles  in  order  to  sensitize  them  to 
the  action  of  alexin.  For  example,  rabbit  red  blood  corpuscles, 
although  strongly  agglutinated  by  heated  hen  serum,  are  no  more 
susceptible  to  the  slight  dissolving  effect  of  normal  guinea-pig  serum 
than  are  normal  rabbit  corpuscles.  The  presence  of  agglutination, 
then,  does  not  indicate  any  particular  sensitivity  to  alexin.  An 
answer  to  these  questions  will  necessitate  further  study. 

CONCLUSIONS. 

I.  The  serum  of  animals  treated  with  defibrinated  blood  from  a 
different  animal  species  shows  active  properties,  consisting  in  the 
agglutination  and  energetic  dissolution  of  corpuscles  similar  to 
those  used  for  injection.     There  is  also  in  certain  instances  a  power 
to  produce  a  precipitate  with  serum  (or  defibrinated  blood)  similar 
to  that  used  in  immunization. 

II.  The  dissolving  action  of  the  active  serum  on  corpuscles  is 
due  to  the  presence  of  two  substances:  one  that  belongs  properly 
to  the  active  serum;  the  other  (alexin)  which  occurs  not  only  in 
active  sera  but  also  in  normal  sera.    The  first  substance  acts  by 
sensitizing  the  corpuscles  to  the  action  of  the  second  substance. 
These  facts  are  strictly  comparable  with  those  already  established 
by  us  for  cholera  serum  and  the  cholera  vibrio. 


AGGLUTINATION  AND   DISSOLUTION.  185 

III.  The  injection  of  an  antihematic  serum  in  a  normal  animal 
of  the  same  species  that  furnished  the  serum  causes  the  appearance 
of  a  similar  property  in  the  serum  of  the  treated  animal.     The 
occurrence  of  this  hemolytic  property  should  be  explained  as  we 
explained  in  1895  the  genesis  of  a  bactericidal  property  in  the  serum 
of  animals  treated  with  cholera  serum.     In  other  words,  this  anti- 
hematic  property  occurs  as  the  result  of  a  meeting  in  the  animal 
body  of  the   characteristic  substance  of  active  serum  with  the 
alexin  which  the  recipient  possessed  before  injection. 

IV.  The  specific  antihematic  substances  characteristic  of  active 
serum  resist  heating  to  55  degrees  and  combine  energetically  with 
the  corpuscles  they  act  on.     Washing  does  not  remove  from  cor- 
puscles their  agglutination  or  their  sensitization  to  alexin  acquired 
by  contact  with  specific  serum.     In  this  respect  the  analogy  to 
bacteria  and  their  specific  sera  also  holds. 

V.  Alexins  from  different  animal  species  although  very  similar 
in  their  action  on  a  given  bacterium  show  distinct  differences  in 
their  action  on  corpuscles.    The  sensitization  of  given  corpuscles 
to  the  action  of  the  alexins  of  certain  animal  species  does  not  neces- 
sarily imply  that  these  corpuscles  are  equally  destroyed  by  all 
alexins. 

VI.  Antihematic  sera  have  also  a  distinct  antitoxic  property; 
they  protect  their  own  corpuscles  against  agglutination  and  dis- 
solution by  the  normal  serum  used  in  injecting  the  animals  that 
have  produced  the  active  serum. 

VII.  There  is  a  close  analogy  between  the  active  substances 
of  specific  sera  and  similar  properties  that  occur  in  normal  sera  in 
respect  to  the  effect  of  heat;  there  is  also  an  analogy  between  the 
substances  affecting  bacteria  and  those  affecting  red  blood  cor- 
puscles in  this  respect.     Heating  to  from  60  to  70  degrees  weakens 
both  the  agglutinating  and  the  sensitizing  property. 

VIII.  Destruction  by  alexin  may  take  place  in  non-agglutinated 
corpuscles.     The  fact  that  corpuscles  are  clumped  by  serum  does 
not   necessarily  imply  that   they  are   sensitized  to  the  action  of 
alexins. 


IX.    HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS,  AND 

THEORIES    CONCERNING    CYTOLYTIC    SERA 

IN    GENERAL.* 

BY  JULES   BORDET. 

I.    ADDITIONAL  IDEAS  CONCERNING  HEMOLYTIC  SERA. 

In  the  present  article  we  propose  to  consider  first  of  all  certain 
novel  facts  concerning  antihematic  sera;f  these  sera,  as  we  know, 
are  obtained  from  animals  that  have  been  treated  with  the  blood 
of  other  species.  We  shall  then  consider  the  principal  properties 
of  an  antitoxin  capable  of  opposing  the  destructive  effect  of  a 
hemolytic  serum  on  red  blood  corpuscles.  And  finally,  we  shall 
take  up  the  theories  that  have  been  offered  to  explain  the  cytolytic 
properties  of  various  immune  sera  and  shall  consider  which  of  these 
theories  are  best  corroborated  by  experimental  facts. 

In  the  following  pages  we  shall  deal  with  a  single  hemolytic  serum 
and  for  this  purpose  have  chosen  the  serum  of  guinea-pigs  treated 
with  rabbit  blood.  This  is  the  serum  that  we  first  used  in  describ- 
ing antihematic  sera.  It  is  easy  to  obtain,  as  guinea-pigs  furnish 
a  very  active  serum  after  receiving  two  or  three  injections  subcuta- 
neously  of  3  to  5  c.c.  of  defibrinated  rabbit  blood.  The  serum  agglu- 
tinates and  destroys  rabbit  blood  corpuscles,  but  has  no  effect  on 
the  corpuscles  of  other  animals. 

We  described  the  properties  of  this  serum  fully  in  a  previous  arti- 
cle (1898) ;  it  seems,  therefore,  scarcely  necessary  to  consider  again 
in  detail  the  effect  of  the  two  substances  present  in  an  antihematic 

*  Les  scrums  he"molytiques,  leurs  antitoxines  et  les  theories  des  scrums  cytoly- 
tiques.  Annales  de  1'Institut  Pasteur,  XIV,  1900,  257. 

f  We  shall  frequently  use  the  expression  "hemolytic  sera,"  or,  preferably,  "hemo- 
toxins"  to  designate  antihematic  sera.  If  the  word  hemotoxin  is  used,  the  anti- 
toxin to  a  hemolytic  serum  should  be  called  antihemotoxin.  These  terms  are 
convenient.  They  were  suggested  by  Metchnikoff.  who  called  a  serum  that  was 
active  against  spermatozoa  a  spermotoxin  and  one  active  against  leucocytes  a 
leucotoxin.  The  expression  "cytolytic"  sera  or  "cytotoxins"  may  be  used  to 
designate  various  immune  sera  that  are  able  to  destroy  bacteria  or  cells  like  red 
blood  cells. 

186 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  187 

serum,  namely,  the  alexin*  or  cellulicidal  substance,  properly  speak- 
ing, destroyed  at  55  degrees ;  and  the  sensitizing  substance  or  specific 
antibody  (preventive  substance)  that  resists  heat  much  better. 
We  must  later  consider  these  facts  in  considering  the  theories 
concerning  bactericidal  and  hemolytic  sera.  We  may  mention 
simply  that  one  of  these  substances,  the  alexin,  is  found,  not  only 
in  immume  serum,  but  also  in  normal  serum.  The  function  of  the 
sensitizing  substance  is  to  render  the  corpuscles  susceptible  to  the 
cytolytic  influence  of  the  alexin.  It  is  scarcely  necessary  to  repeat 
in  the  following  experiments  that  heating  to  55  degrees  for  half  an 
hour  destroys  the  cytolytic  activity  of  the  sera  employed. 

1.  The  dissolving  properties  of  different  alexins  in  the  presence 
of  the  sensitizing  substance. 

We  know  that  our  hemotoxin  loses  its  hemolytic  activity  when 
heated  to  55  degrees  and  that  the  addition  of  normal  guinea-pig 
serum  restores  its  primitive  energy;  this  added  serum,  however,  is 
only  slightly  cellulicidal  in  itself.  The  same  result  may  be  obtained 
by  using  normal  rabbit  serum  instead  of  normal  guinea-pig  serum : 
we  have  already  mentioned  this  remarkable  fact  and  shall  frequently 
return  to  it,  that  the  alexin  in  normal  rabbit  serum  which  is  wholly 
inoffensive  for  its  proper  corpuscles  becomes  strongly  hemolytic 
for  them  when  associated  with  a  sensitizing  substance,  f  We  have 
then  two  different  alexins,  normal  rabbit  serum  and  normal  guinea- 
pig  serum,  either  of  which  can  destroy  sensitized  rabbit  corpuscles. 
Do  the  alexins  from  other  animals  act  in  the  same  way?  In  other 
words,  does  the  sensitizing  substance  increase  the  destructive  prop- 
erty of  various  normal  sera  considerably? 

An  experiment  to  answer  this  question  shows  that  sensitized 
rabbit  corpuscles  are  rapidly  dissolved  by  the  normal  serum  of  the 
rat,  the  goat  and  the  dog,  none  of  which,  however,  without  the  addi- 
tion of  the  sensitizing  substance  is  more  than  faintly  destructive. 

*  We  think  it  quite  unnecessary  to  replace  the  word  alexin  by  any  other;  this 
word  was  introduced  some  time  ago  by  Buchner  and  has  been  frequently  employed 
by  numerous  observers  and  become  a  customary  term.  It  matters  little  what 
word  is  used  provided  its  significance  is  understood. 

t  We  may  mention  once  for  all  that  by  the  word  sensitizing  substance  we  mean 
a  hemolytic  serum  heated  to  55  degrees  and  so  deprived  of  alexin  and  containing 
only  the  sensitizing  substance  or  specific  antibody. 


188  STUDIES  IN  IMMUNITY. 

The  presence  of  the  sensitizing  substance  then  increases  the  herao- 
lytic  power  of  these  sera  distinctly.  It  also  increases  to  a  less  extent 
the  activity  of  normal  pigeon  serum.  The  normal  sera  of  the 
hen  and  the  goose  are  in  themselves  very  hemolytic  for  rabbit  cor- 
puscles and  the  addition  of  sensitizing  substance  does  not  appear 
to  increase  their  properties  to  any  great  extent. 

It  is  to  be  noted  that  guinea-pig  serum  is  the  one  that  affects 
corpuscles  subjected  to  sensitizing  substance  most  actively;  this 
serum  is  much  more  effective  than  rabbit  serum.  Very  large  doses 
of  rabbit  serum  are  necessary  to  destroy  sensitized  rabbit  corpuscles 
when  a  small  dose  of  sensitizing  substance  is  used.*  An  analogous 
fact  has  recently  been  observed  by  Buchner.f  According  to  this 
observer  bovine  red  blood  corpuscles  treated  with  an  appropriate 
sensitizing  substance  from  the  rabbit  are  easily  destroyed  by  nor- 
mal dog  serum.  In  a  general  way  these  facts  have  been  known  for 
some  time.  Five  years  ago  we  mentioned  an  analogous  series  of 
facts  in  showing  that  cholera  vibrios  treated  with  a  little  anti- 
cholera  sensitizing  substance  (that  is  to  say,  immune  serum  from 
a  goat  heated  to  55  degrees)  were  changed  in  vitro  into  roundish 
granules  when  subjected  to  normal  serum  from  the  guinea-pig, 
rabbit,  rat,  man  or  goat.  More  recently  we  added  to  this  list  sera 
of  the  dog,  hen  and  pigeon. $  In  these  instances  the  sensitized 
vibrios  are  affected  by  the  alexins  present  in  normal  sera. 

The  same  facts  hold  for  corpuscles. 

For  completeness  we  must  recall  an  already  published  observa- 
tion. A  year  ago§  we  first  mentioned  that  sensitized  corpuscles 
are  not  indiscriminately  dissolved  by  any  normal  serum,  or,  in 
other  words,  by  every  alexin.  For  example,  sensitized  hen  cor- 
puscles (hemolytic  serum  from  the  rabbit  heated  to  55  degrees) 
are  readily  dissolved  by  several  normal  sera,  but  remain  quite  intact 
in  normal  hen  serum.  This  seems  then  to  be  a  certain  contradiction 
to  the  statement  that  sensitized  rabbit  corpuscles  are  destroyed  by 
rabbit  serum.  When  we  mentioned  this  apparent  contradiction  a 

*  If  the  energy  of  the  sensitizing  substance  is  slightly  diminished,  small  doses 
of  rabbit  serum  will  frequently  fail  to  dissolve  the  corpuscles,  although  under  the 
same  conditions  normal  guinea-pig  serum  is  still  very  effective. 

t  Munchener  medicinische  Wochenschrift,  1900. 

t  See  p.  180. 

§  See  p.  173. 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  189 

year  ago  we  said  that  the  modification  of  the  corpuscle  by  the  sen- 
sitizing substance  might  be  likened,  as  Fischer  has  done  for  diastases, 
to  a  change  in  a  lock  so  that  keys  that  could  not  previously  open  it 
were  enabled  to  do  so.  In  such  a  way  certain  alexins  would  " enter" 
a  sensitized  corpuscle  and  destroy  it;  and  other  slightly  different 
alexins  could  not  attack  it.  There  must  exist,  then,  certain  suitable 
relations  between  the  sensitizing  substance  and  the  alexin  em- 
ployed in  order  to  bring  about  a  destruction  of  the  corpuscles.  We 
shall  not  insist  further  on  these  conceptions,  which  have,  indeed, 
been  made  use  of  by  Wassermann  in  a  recent  article.* 

2.  The  identity  in  a  given  serum  of  the  bacteriolytic  and  the 
hemolytic  alexin. 

A  normal  serum,  say  from  the  guinea-pig,  in  presence  of  an  anti- 
cholera  sensitizing  substance  attacks  vibrios  and  causes  their  gran- 
ular transformation.  The  same  normal  serum  destroys  red  blood 
corpuscles  in  presence  of  a  hemolytic  sensitizing  substance.  When 
the  normal  serum  is  heated  to  55  degrees  it  has  no  longer  either 
bacteriolytic  or  hemolytic  effect  even  in  the  presence  of  appropriate 
sensitizing  substances.  This  fact  is  explained  by  saying  that  heat- 
ing to  55  degrees  destroys  the  alexin,  which  causes  cytolysis.f  The 
question  arises  as  to  whether  normal  guinea-pig  serum  contains  a 
single  alexin  that  is  both  hemolytic  and  bacteriolytic,  or  several 
alexins,  one  or  several  of  which  can  destroy  bacteria  and  the  other, 
or  several  of  which,  preferably  attack  red  blood  corpuscles.  This 
question  is  open  to  experimental  proof. 

When  normal  cholera  vibrios  are  mixed  with  normal  guinea-pig 
serum  they  are  not  destroyed,  but  are  only  slightly  affected,  since, 
not  being  sensitized,  the  alexin  cannot  react  with  them.J  If  to  such 
a  mixture  sensitized  corpuscles  are  added  (that  is  to  say,  rabbit 
corpuscles  plus  hemotoxin  heated  to  55  degrees)  we  find  that  they 
are  at  once  destroyed,  which  proves  that  the  normal  cholera  vibrios 
have  neither  transformed  nor  fixed  the  alexin  necessary  for  destruc- 
tion of  corpuscles. 

We  may  repeat  this  experiment  with  a  modification.     We  may 

*  Wassermann,  Deutsche  medicinische  Wochenschrift,  1900. 

t  Heating  to  55  degrees  destroys  the  cytolytic  property,  not  only  of  guinea-pig 
serum,  but  also  of  sera  from  the  rabbit,  rat,  goat,  hen,  etc. 

|  We  already  mentioned  some  time  ago  (see  p.  61,  1895)  that  normal  serum 
destroys  few  vibrios  unless  they  be  very  attenuated. 


190  STUDIES  IN   IMMUNITY. 

add  to  the  same  dose  of  normal  guinea-pig  serum  cholera  vibrios 
sensitized  with  cholera  serum  instead  of  normal  vibrios.  Under 
such  conditions  the  sensitized  vibrios  show  rapid  granular  trans- 
formation. If  we  then  add  to  the  mixture,  as  we  did  in  the  first 
experiment,  sensitized  corpuscles  we  find  that  they  remain  indefi- 
nitely intact.  From  this  experiment  we  conclude  that  the  alexin 
necessary  to  destroy  the  corpuscles  was  used  up  before  they  were 
introduced.  In  the  presence  of  the  cholera  sensitizing  substance 
the  alexin  reacted  with  the  vibrios  and,  in  transforming  them  into 
granules,  was  fixed  by  them.  Consequently  the  alexin  that  unites 
with  sensitized  vibrios  and  alters  them  is  identical  with  the  one  that 
produces  hemolysis. 

EXPERIMENT.  An  emulsion  of  vibrios  is  prepared  by  suspending  a  24-hour 
agar  culture  in  5  c.c.  of  salt  solution.  The  cholera  sensitizing  substance  employed 
is  from  a  vaccinated  rabbit  and  has  been  heated  to  55  degrees.  As  a  control  for 
this  active  serum  normal  rabbit  serum,  also  heated  to  55  degrees,  is  used. 

Mixture  A.  0.5  c.c.  of  normal  guinea-pig  serum  plus  0.3  c.c.  of  cholera  sensi- 
tizing substance  plus  0.5  c.c.  of  vibrio  emulsion. 

Mixture  B.  0.5  C;C.  of  normal  guinea-pig  serum,  0.3  c.c.  of  normal  rabbit 
serum  (55  degrees)  plus  0.5  c.c.  of  vibrio  emulsion. 

Mixture  C.     Identical  with  A  but  containing  no  vibrios. 

An  hour's  contact  is  allowed  and  there  is  then  added  to  each  mixture  0.2  of  a 
cubic  centimeter  of  hemosensitizing  substance  (55  degrees)  plus  2  drops  of  washed 
rabbit  blood.* 

The  results  of  the  experiments  are  as  follows:  the  corpuscles  are  rapidly 
destroyed  in  B  and  C,  but  remain  intact  in  A. 

A  similar  experiment  with  the  cells  (bacteria  and  corpuscles)  used 
in  the  inverse  order  gives  a  similar  result.  We  know  from  the  work 
of  Ehrlich  and  Morgenroth  as  well  as  from  our  own  that  an  alexin 
that  does  not  destroy  given  red  blood  cells  is  not  fixed  by  them,  but 
is,  on  the  contrary,  fixed  by  cells  that  it  can  dissolve. 

An  alexin  that  under  normal  circumstances  has  no  effect  on  a 
given  species  of  corpuscles  and  therefore  is  not  fixed  by  them  is 
fixed  by  these  same  corpuscles  when  they  have  been  treated  with 
a  suitable  sensitizing  substance.  The  fixation  of  the  alexin  by 

*  In  the  majority  of  experiments  we  use  previously  washed  blood  instead  of 
simple  defibrinated  blood.  This  washing  gives  us  rabbit  blood  corpuscles  without 
the  addition  of  the  serum  present  in  defibrinated  blood.  The  corpuscles  are 
washed  by  adding  a  relatively  large  amount  of  salt  solution  to  a  small  amount  of 
defibrinated  blood,  centrifugalizing,  and  decanting  the  supernatant  fluid. 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  191 

sensitized  corpuscles  is  the  important  fact  that  Ehrlich  and  Morgen- 
roth  were  the  first  to  show  experimentally.*  This  fact  would 
seem  to  be  a  remarkable  confirmation  of  the  idea  that  we  established 
several  years  ago,  namely,  that  under  the  influence  of  a  suitable 
sensitizing  substance  (preventive  substance)  the  animal  body 
directs  this  bactericidal  (or  cellulicidal)  power  particularly  against 
a  given  cell. 

Normal  rabbit  corpuscles  remain  intact  when  mixed  with  fresh 
normal  guinea-pig  serum  and  the  alexin  is  not  absorbed.  If  we 
subsequently  add  sensitized  cholera  vibrios  to  this  mixture,  they  soon 
show  a  granular  transformation,  which  proves  that  the  fluid  con- 
tains free  alexin.  But  if  in  another  tube  we  mix  normal  guinea- 
pig  serum  with  rabbit  corpuscles  treated  with  a  hemosensitizing 
substance,  they  are  destroyed,  absorb  the  alexin,  and  sensitized 
vibrios  subsequently  added  remain  normal  and  show  no  transfor- 
mation. 

EXPERIMENT.  An  emulsion  of  cholera  vibrios  is  made  by  suspending  an  agar 
culture  in  10  c«c.  of  salt  solution.  About  a  third  of  this  volume  of  cholera  serum 
from  the  rabbit,  heated  to  55  degrees  for  half  an  hour,  is  then  added.  This  forms 
an  emulsion  of  sensitized  vibrios.  The  blood  used  is  washed  rabbit  blood. 

Mixture  A.  0.3  of  a  cubic  centimeter  of  rabbit  blood  plus  0.6  of  a  cubic 
centimeter  of  hemolytic  serum  (55  degrees)  plus  0.3  c.c.  normal  guinea-pig  serum. 
Complete  and  rapid  hemolysis  occurs. 

Mixture  B.  Identical  with  A  with  the  exception  that  0.6  of  a  cubic  centimeter 
of  normal  guinea-pig  serum  (55  degrees)  is  used  instead  of  a  specific  serum.  No 
hemolysis. 

Mixture  C.  Identical  with  A  but  with  guinea-pig  blood,  0.3  of  a  cubic  centi- 
meter, replacing  the  rabbit  blood.  In  this  mixture  there  is  no  hemolysis,  as  the 
guinea-pig  corpuscles  are  not  affected  by  fresh  guinea-pig  serum  and  heated 
hemolytic  serum. 

In  1  to  2  hours  0.1  of  a  cubic  centimeter  of  the  emulsion  of  sensitized 
vibrios  is  added  to  each  mixture.  The  mixtures  are  then  placed  in  the  incubator 
for  an  hour  with  the  following  results:  in  mixture  A,  where  hemolysis  took  place, 
the  vibrios  undergo  no  transformation.  There  is  complete  granular  transforma- 
tion in  mixtures  B  and  C,  in  which  the  corpuscles  were  intact. 

3.  The  fixation  by  corpuscles  of  the  active  substances  of  hemolytic 
serum.  The  action  of  the  stromata. 

Rabbit  corpuscles  when  mixed  with  our  hemolytic  serum  pre- 
viously heated  to  55  degrees  remain  intact,  with  the  exception  of 
being  agglutinated.  Under  these  conditions  they  fix  the  sensitizing 

*  See  "Collected  Studies  on  Immunity,"  Ehrlich-Bolduan,  Wiley  &  Co.,  p.  1. 


192  STUDIES  IN   IMMUNITY. 

substance.*  The  same  corpuscles,  when  mixed  with  either  fresh 
hemolytic  serum  or  a  mixture  of  fresh  normal  serum  plus  heated 
hemolytic  serum,  are  destroyed.  Under  these  conditions  it  is  easy 
to  demonstrate  that  they  have  fixed  both  the  sensitizing  substance 
and  the  alexin. 

We  may  now  consider  what  part  of  the  corpuscles  absorbs  the 
active  substances.  If  corpuscles  are  placed  in  distilled  water  they 
lose  their  hemoglobin,  as  we  know,  and  are  reduced  to  transparent 
stromata  floating  in  a  red  fluid.  We  may  determine  whether  the 
hemotoxic  substances  are  fixed  by  the  substances  dissolved  in  the 
laky  fluid  or  by  the  stromata  suspended  in  it.  For  such  an  experi- 
ment the  stromata  are  separated  from  the  fluid  in  which  they  are 
suspended. 

We  take  3  c.c.  of  defibrinated  rabbit  blood,  and  preferably  of  blood 
that  has  been  washed  in  salt  solution.  To  this  is  added  15  c.c.  of 
distilled  water  and  a  very  red  and  nearly  transparent  fluid  is  ob- 
tained. Microscopically  the  stromata  are  scarcely  visible,  are  very 
translucent,  with  a  full  round  outline,  and  apparently  are  filled  with 
water.  To  this  laked  red  fluid  is  added  1  c.c.  of  distilled  water  con- 
taining 0.0975  of  a  gram  of  sodium  chloride.  The  addition  of  this 
salt  solution  restores  the  laked  fluid  to  a  tonicity  corresponding  to 
normal  salt  solution.  This  addition  of  salt  solution  produces  an 
immediate  clouding  of  the  nearly  transparent  fluid.  This  cloud- 
iness settles  out  in  whitish  flecks  which  gradually  fall  to  the  bottom 
of  the  tube,  leaving  a  clear  supernatant  fluid.  Microscopical  exam- 
ination shows  these  flecks  to  be  composed  of  agglutinated  stromata. 
These  stromata  have  lost  their  very  transparent  and  regularly 
rounded  form,  have  become  more  visible,  and  are  flattened  out  into 
thin  concave  disks.  When  seen  in  cross  section  they  seem  to  have 
undergone  a  veritable  plasmolysis  and  it  would  seem  as  if  the 
stromata  were  simply  inclosing  membranes  or  veritable  closed 
sacs  that  swell  in  hypotonic  fluids,  and  contract  and  flatten  out  in 
more  concentrated  solutions. 

However  that  may  be,  the  fluid  is  easily  separable  into  two  parts, 

either  by  deposition  or,  better,  by  centrifugalization :  an  upper  part, 

which  is  absolutely  limpid,  with  no  microscopically  visible  elements; 

and  the  other  reddish,  opaque,  and  including  large  numbers  of  sus- 

*  As  we  have  already  previously  noted,  they  also  fix  the  agglutinin. 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  193 

pended  stromata.*  To  equal  amounts  of  these  two  fluids  equal 
amounts  of  sensitizing  substance  and  of  alexin  (from  a  normal 
guinea-pig)  are  added.  The  alexin  should  not  be  in  too  large 
amount.  The  mixtures  are  then  shaken  and,  after  a  certain  time, 
well-sensitized  rabbit  corpuscles  are  added  to  each  fluid.  It  is 
found  that  these  corpuscles  are  not  hemolyzed  or  are  only  slightly 
hemolyzed  in  the  fluid  containing  the  stromata,  but,  on  the  contrary, 
are  rapidly  dissolved  in  the  limpid  red  fluid.  From  this  experi- 
ment it  may  be  concluded  that  the  property  possessed  by  the  red  blood 
corpuscles  of  absorbing  alexin  in  presence  of  the  sensitizing  substance 
is  due  to  their  stromata. 

For  another  experiment  a  suitable  dose  of  sensitizing  substance 
(that  is,  hemolytic  serum  heated  to  55  degrees)  may  be  added  to  the 
two  fluids.  After  shaking  and  allowing  to  stand  for  a  time  the 
second  mixture  is  centrifugalized  to  separate  out  the  stromata,  and 
the  supernatant  fluid  is  decanted.  It  is  then  demonstrable  that 
this  fluid  contains  no  sensitizing  substance,  since  the  subsequent 
addition  of  red  blood  cells  and  normal  guinea-pig  serum  gives  no 
hemotysis.  The  property  of  absorbing  the  sensitizing  substance  also 
belongs,  then,  to  the  stromata. 

In  yet  another  experiment  it  may  be  shown  that  the  stromata 
absorb  alexin  when  the  sensitizing  substance  is  present,  whereas 
they  do  not  fix  it  if  the  latter  substance  is  absent.  For  this  experi- 
ment a  thick  emulsion  of  stromata  is  prepared  to  which  a  large 
amount  of  salt  solution  is  added.  The  mixture  is  centrifugalized 
and  the  limpid  pinkish  supernatant  fluid  decanted.  The  washing 
with  salt  solution  is  repeated  until  the  emulsion  of  stromata  is  quite 
white  and  free  from  hemoglobin.  The  emulsion  is  then  divided 
into  two  equal  parts,  to  each  of  which  the  same  dose  of  alexin  (serum 
of  a  normal  guinea-pig)  is  added.  To  the  first  mixture  a  small 
amount  of  sensitizer  is  then  added  and  to  the  second  the  same 
amount  of  normal  guinea-pig  serum  also  heated  to  55  degrees.  It 
may  be  demonstrated  in  the  usual  manner  that  in  the  first  mixture 
the  alexin  has  disappeared  from  the  fluid  by  fixation  on  the 
stromata;  there  is  no  absorption  of  the  alexin  in  the  second  fluid 
containing  no  sensitizing  substance. 

*  It  is  well  to  determine,  of  course,  that  there  are  no  intact  corpuscles  remaining 
among  these  stromata. 


194  STUDIES  IN  IMMUNITY. 

In  brief,  then,  red  blood  corpuscles  owe  their  characteristic 
absorbing  properties  to  their  stromata.  Is  the  property  in  rabbit 
corpuscles  of  producing  a  hemolytic  power  when  injected  in  guinea- 
pigs  also  due  to  their  stromata?  To  answer  this  question  stromata 
that  have  been  freed  from  all  soluble  substances  by  carefully  wash- 
ing in  salt  solution  are  obtained  and  injected  into  guinea-pigs. 
Each  guinea-pig  receives  the  stromata  from  4  to  5  c.c.  of  defibri- 
nated  blood.  Corresponding  guinea-pigs  are  injected  with  a  lim- 
pid reddish  fluid  containing  the  soluble  substances  obtained  from 
corpuscles  by  treating  them  with  distilled  water  and  subsequently 
adding  salt.  This  fluid  is  then  centrifugalized  to  deprive  it  of  sus- 
pended stromata.  After  injection  lasting  for  three  weeks  it  is  found 
that  guinea-pigs  injected  with  stromata  furnish  an  active  hemolytic 
serum;  those  that  receive  the  limpid  fluid  containing  hemoglobin 
have  a  serum  like  that  of  normal  guinea-pigs. 

Corpuscles  fix  the  active  substances  of  hemolytic  serum.  Can 
the  nature  of  this  phenomenon  of  fixation  be  more  exactly  deter- 
mined? Is  it  a  question  of  true  chemical  combination  by  the  union 
of  certain  elements  of  the  red  blood  cells  with  the  active  principles, 
and  does  it  take  place  according  to  fixed  proportions?  Or  should 
we  consider  the  fixation  by  corpuscles  as  more  like  a  dyeing  phe- 
nomenon? A  substance  to  be  dyed,  as  we  know,  absorbs  extremely 
varying  amounts  of  the  dye,  so  as  to  take  shades  of  varying  inten- 
sity. The  amount  of  dye  that  is  fixed  is  subject  to  wide  variations; 
chemical  reactions,  properly  speaking,  on  the  other  hand,  are 
characterized  by  a  reaction  in  definite  proportions. 

We  shall  not  consider  this  difficult  problem  fully,  as  we  have  not 
as  yet  sufficient  data,  but  a  single  experiment  may  be  given  which 
may  prove  important  in  future  studies  of  this  subject. 

A  small  amount  of  defibrinated  rabbit  blood  (for  example,  0.1 
of  a  cubic  centimeter)  is  added  to  a  certain  amount  of  fresh  hemo- 
lytic serum  (for  example,  0.4  of  a  cubic  centimeter).  The  corpuscles 
are  rapidly  destroyed.  The  hemolysis  although  still  rapid  is  some- 
what slower  if  a  large  dose  of  blood  is  added  (say  0.3  or  0.4  of  a 
cubic  centimeter).  The  destruction  of  corpuscles  naturally  enough 
varies  in  rapidity  with  the  amount  of  blood  added  to  the  hemotoxin. 
And  yet  the  serum  that  is  used  can  destroy  completely  and  relatively 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  195 

rapidly  an  equal  or  even  a  slightly  superior  volume  of  rabbit  blood. 
For  example  0.5  of  a  cubic  centimeter  of  blood  added  to  0.4  of  a 
cubic  centimeter  of  serum  gives  a  complete  destruction  of  all  the 
corpuscles  in  about  an  hour. 

In  this  latter  experiment  we  have  supposed  an  instance  in  which 
the  amount  of  blood  (0.5  of  a  cubic  centimeter)  was  added  all  at  once 
to  the  0.4  of  a  cubic  centimeter  of  serum.  But  the  blood  may  also 
be  added  in  fractions;  we  may  add  to  the  0.4  of  a  cubic  centimeter  of 
hemo toxin,  first  0.2  of  a  cubic  centimeter  of  blood,  and  later  0.1  of  a 
cubic  centimeter  and,  after  an  hour  or  two,  a  third  0.2  of  a  cubic 
centimeter.  In  other  words,  we  determine  whether  a  given  amount 
of  serum  is  uniformly  able  to  destroy  the  same  number  of  corpuscles 
whether  they  be  added  all  at  once  or  in  divided  doses.  As  a  matter 
of  fact  it  is  found  that  the  dissolving  property  of  serum  is  rapidly 
exhausted  if  the  blood  is  introduced  gradually  in  small  amounts, 
particularly  when  the  time  intervals  between  the  doses  are  some- 
what prolonged.  We  find  that  0.4  of  a  cubic  centimeter  of  fresh 
hemolytic  serum  —  an  amount  capable  of  destroying  completely 
at  least  0.5  of  a  cubic  centimeter  of  blood  when  added  at  once  —  dis- 
solves no  more  than  0.2  of  a  cubic  centimeter  of  blood  if  the  cor- 
puscles are  added  to  the  serum  in  divided  doses.  The  first  doses 
introduced  are  well  dissolved  until  the  total  amount  of  blood  added 
is  about  0.2  of  a  cubic  centimeter,  but  subsequently  added  corpuscles 
remain  intact.  At  least  twice  as  much  blood  then  may  be  dis- 
solved when  added  to  serum  all  at  once  as  when  added  in  divided 
doses.  It  is  evident  that  in  the  latter  instance  the  first  corpuscles 
added  become  in  some  manner  supersaturated  with  the  active  sub- 
stance and  exhaust  the  fluid  so  that  it  becomes  inactive  for  other 
corpuscles.  In  other  words,  the  first  corpuscles  absorb  more  active 
substances  than  is  necessary  for  their  dissolution. 

This  seems  to  us  to  favor  the  conception  that  the  absorption  of 
active  principles  by  the  corpuscles  should  be  compared  to  a  dyeing 
phenomenon.  In  such  phenomena,  as  we  know,  the  substances  to 
be  dyed  absorb  varying  amounts  of  the  dye  under,  conditions  that 
are  quite  similar  to  those  just  outlined  for  corpuscles. 

If  we  pour  10  c.c.  of  a  diluted  rather  pale  solution  of  methyl  violet 
in  a  crystallizing  dish,  a  piece  of  filter  paper  placed  in  it  becomes 
colored.  It  soon  takes  on  a  uniform  pale  tint,  while  the  fluid,  as  it 


196  STUDIES  IN   IMMUNITY. 

is  exhausted,  becomes  decolorized.  Let  us  now  take  the  same 
amount  of  coloring  fluid  and  a  piece  of  filter  paper  of  exactly  the 
same  size  as  before.  Instead  of  plunging  this  entire  piece  of  paper 
into  the  fluid  we  cut  it  up  into  pieces.  When  we  place  the  first 
piece  of  paper  in  the  fluid  it  takes  on  a  very  dark  shade  and  de- 
prives the  coloring  fluid  of  a  good  deal  of  its  color.  We  then  add 
a  second  fragment  of  paper,  and,  after  another  interval,  a  third. 
These  latter  pieces  take  only  a  very  faint  tinge  owing  to  the  more 
or  less  complete  decolorization  of  the  fluid.  The  fluid  soon  becomes 
entirely  decolorized  and  the  last  fragments  added  remain  quite  white. 
By  analogy  we  may  consider  that,  in  the  first  instance,  when  red 
blood  corpuscles  are  added  in  a  single  dose  they  become  sus- 
ceptible to  a  loss  of  hemoglobin,  although  they  only  " stain  faintly" 
with  the  active  principles,  but  that  in  the  latter  conditions,  when 
added  in  divided  doses,  they  absorb  a  much  larger  dose  of  these 
substances  and  so  exhaust  the  serum  and  prevent  the  destruction 
of  subsequently  added  corpuscles. 

It  is  difficult  to  understand  just  how  alexin  destroys  sensitized 
corpuscles  and  causes  their  hemoglobin  to  pass  out.  We  may  men- 
tion a  fact  that  may  have  some  bearing  on  researches  along  this 
line.  We  add  defibrinated  rabbit  blood  to  a  rather  small  dose  of 
hemotoxin  and  wait  until  hemolysis  is  complete.  At  the  same  time 
we  add  a  corresponding  amount  of  normal  guinea-pig  serum  to  the 
same  amount  of  blood,  in  which  case  the  corpuscles  remain  intact. 
We  then  add  to  each  mixture  a  large  amount  of  distilled  water,  which 
will  destroy  the  corpuscles  in  the  second  mixture  as  well.  We  have 
then  two  fluids;  in  the  first  the  corpuscles  have  been  destroyed  by 
active  serum  and  in  the  second  by  distilled  water.  To  each  of  these 
fluids  we  then  add  sufficient  salt  to  render  them  isotonic.  In  the 
mixture  in  which  the  corpuscles  were  dissolved  by  distilled  water 
the  stromata  show  energetic  plasmolysis  and  retract.  In  the  other 
fluid  containing  hemotoxin  the  stromata  keep  their  primitive 
rounded  form  and  apparently  do  not  show  any  effect  from  the 
increased  concentration.  It  would  seem  as  if  the  alexin  from  the 
active  serum  had  destroyed  or  digested  something  in  the  corpuscles 
that  has  to  do  with  the  action  of  plasmolysis  and  phenomena  of  osmosis. 
The  diffusion  of  soluble  hemoglobin  would  perhaps  be  the  result  of 
such  destruction. 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  197 

II.    THE  ANTITOXIN  TO  HEMOLYTIC  SERUM. 

A  serum  with  antitoxic  properties  for  the  hemotoxin  that  we 
have  been  considering  in  the  preceding  pages  is  easily  obtained. 
It  is,  in  other  words,  a  substance  capable  of  protecting  rabbit  cor- 
puscles from  the  destructive  power  of  our  hemolytic  serum.*  This 
hemolytic  serum,  as  already  shown,  is  toxic  for  the  rabbit.  When 
injected  in  doses  of  5  c.c.  or  thereabouts  intravenously  it  kills  almost 
instantaneously.  At  autopsy  clots  bathed  with  reddish  serum  are 
found  in  the  heart  and  large  vessels,  which  indicates  that  hemolysis 
has  taken  place  in  vivo.  Disseminated  hemorrhagic  effusions  are 
also  frequently  found  in  the  kidney  and  muscles,  particularly  in  the 
psoas  muscle.  The  hemotoxin  when  injected  subcutaneously  into 
rabbits  in  a  small  dose  produces  no  severe  effect  and  animals  treated 
in  this  manner  acquire  an  antitoxic  power  in  their  serum. 

The  hemotoxin  is  injected  two  or  three  times,  at  intervals  of 
15  days,  in  a  dose  of  from  2  to  3  c.c.  The  rabbits  are  then  bled 
12  to  15  days  after  the  last  injection. 

The  antitoxic  (antihemolytic  or  antihemotoxic)  serum  obtained 
by  this  relatively  short  treatment  is  not,  to  be  sure,  very 
powerful;  it  is  sufficiently  so,  however,  to  permit  a  study  of  its 
properties. 

The  simplest  experiment  to  demonstrate  its  antitoxic  property 
consists  in  adding  a  relatively  large  dose  of  freshly  obtained  anti- 
toxic serum  to  a  small  amount  of  fresh  hemolytic  serum.  To  this 
mixture  is  then  added  a  small  amount  of  rabbit  blood.  As  a  control 
a  mixture  of  hemolytic  serum,  normal  rabbit  serum  and  rabbit 
blood  is  made  at  the  same  time  and  in  the  same  proportions. 

If  the  dose  of  antitoxin  is  sufficient,  no  dissolution  of  corpuscles 
takes  place  in  the  first  mixture.  In  the  second  mixture,  on  the 
contrary,  containing  the  same  dose  of  normal  rabbit  serum  instead 
of  antitoxic  serum,  the  corpuscles  are  destroyed. 

In  such  an  experiment  a  considerably  greater  dose  of  antitoxin 
than  of  hemolytic  serum  (10  to  20  times)  must  be  used  to  protect 

*  We  have  already  shown  (see  p.  175)  that  an  antihemolytic  antitoxin  similar 
to  that  described  by  Camus  and  Gley  and  Kossel  for  eel  serum  may  be  obtained. 
The  serum  that  we  described  opposed  the  hemolytic  effect  of  hen  serum. 

Metchnikoff  has  recently  described  an  antispermatoxin  (Annales  de  1'Institut 
Pasteur,  1900,  No.  1). 


198  STUDIES  IN  IMMUNITY. 

the  corpuscles  properly.  It  will  be  recalled  that  in  these  experi- 
ments we  employed  an  active  hemotoxin  from  guinea-pigs  that  have 
received  three  injections  of  4  to  5  c.c.  of  rabbit  blood. 

We  may  note  at  once  that  the  antitoxic  serum  is  in  reality  more 
active  than  would  seem  from  this  preliminary  experiment;  by  a 
very  simple  procedure  it  may  be  made  to  neutralize  much  larger 
quantities  of  hemolytic  serum  perfectly.  If  the  serum  is  heated 
to  55  degrees  it  is  found  that  three  or  even  two  volumes  of  anti- 
toxic serum  will  completely  neutralize  one  volume  of  fresh  hemo- 
lytic serum. 

As  we  shall  presently  see,  the  explanation  of  this  fact  is  simple. 
When  the  antitoxic  serum  is  fresh  it  contains  an  excess  of  rabbit 
alexin,  which,  in  the  presence  of  the  sensitizing  substance,  is  dan- 
gerous for  the  rabbit  corpuscles.  In  order,  therefore,  to  demonstrate 
its  maximum  protective  power  one  should  eliminate  this  harmful 
substance  by  heating  the  serum  to  55  degrees. 

That  the  antitoxic  serum  does  possess  an  alexin  capable  of  de- 
stroying rabbit  corpuscles  when  the  sensitizing  substance  is  present 
as  well  as  normal  rabbit  serum,  is  easily  demonstrable.  On  mixing 
hemolytic  serum  heated  to  55  degrees  with  equal  parts  of  fresh  anti- 
toxic serum  and  adding  a  small  amount  of  rabbit  blood  we  find  that 
the  corpuscles  are  dissolved.  This  destruction  is  due  to  the  alexin 
in  the  antitoxin  since  there  is  none  in  the  hemolytic  serum.  If  we 
should  add  too  great  a  dose  of  antitoxin  to  the  sensitizing  substance, 
there  would  be  no  hernolysis.  For,  as  we  shall  see,  the  sensitizing 
substance  will  have  been  neutralized  and  under  these  conditions 
the  alexin  in  the  antitoxin  can  have  no  harmful  effect  on  the 
corpuscles.  In  the  presence  of  the  sensitizing  substance,  then,  a 
small  dose  of  antitoxin  destroys  the  corpuscles  but  a  large  dose 
protects  them. 

In  order,  then,  to  study  the  properties  of  the  antitoxin  to  best 
advantage  it  is  better  to  heat  the  serum  to  55  degrees  as  a  general 
procedure. 

Antisensitizing  substance  and  anti-alexin. — We  know  that  hemo- 
lytic serum  destroys  rabbit  corpuscles  owing  to  the  action  of  two 
distinct  substances :  the  specific  sensitizing  substance  (antibody  or 
preventive  substance)  and  the  alexin.  This  latter  substance,  which 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  199 

occurs  in  normal  rabbit  serum,  attacks  rabbit  corpuscles  treated 
with  the  sensitizing  substance. 

Since  hemolytic  serum  has  two  active  substances,  both  of  which 
must  be  present  to  produce  hemolysis,  it  is  at  once  evident  that 
the  antitoxin  may  protect  corpuscles  by  neutralizing  either  one 
of  these  substances.  Which  substance,  then,  does  the  antitoxin 
neutralize?  Or  does  it  affect  them  both  simultaneously  ? 

The  following  experiments  that  we  are  about  to  describe  answer 
this  question;  but  we  may  state  the  conclusion  at  once,  namely, 
that  antitoxic  serum  neutralizes  both  the  sensitizing  substance  and  the 
alexin  of  hemolytic  serum. 

In  order  to  demonstrate  the  neutralizing  effect  of  the  antitoxin 
for  the  sensitizing  substance  we  make  use  of  the  fact  that  rabbit 
corpuscles  affected  by  the  sensitizing  substance  may  be  destroyed, 
not  only  by  normal  guinea-pig  serum,  but  also  by  normal  rabbit 
serum.  Normal  rabbit  serum  is,  of  course,  under  ordinary  conditions 
quite  harmless  for  rabbit  corpuscles  and  it  therefore  serves  as  an 
excellent  indicator  of  the  presence  of  the  sensitizing  substance  in  a 
fluid.  It  may  be  shown,  moreover,  by  a  suitable  experiment  that 
our  antitoxin  from  the  rabbit  has  no  neutralizing  effect  on  rabbit 
alexin.  Consequently,  if,  in  a  suitably  chosen  mixture  of  heated 
hemolytic  serum,  heated  antitoxic  serum,  and  fresh  normal  rabbit 
serum,  subsequently  introduced  corpuscles  remain  intact,  we  may 
conclude  that  the  sensitizing  substance  of  the  heated  hemolytic 
serum  has  been  neutralized  by  the  antitoxin.  The  control  naturally 
is  composed  of  identical  doses  of  each  substance  with  normal  rabbit 
serum  heated  to  55  degrees  replacing  the  antitoxic  serum.  In  such 
a  control  mixture  corpuscles  treated  with  sensitizing  substance  are 
easily  destroyed  by  the  alexin  present  in  the  normal  rabbit  serum. 

From  such  experiments  we  learn  that  the  antisensitizing  function 
of  our  antitoxin  is  only  slightly  developed,  and  at  least  15  parts  of 
antitoxin  are  necessary  to  neutralize  the  sensitizing  substance  in 
1  part  of  heated  hemolytic  serum.  The  intensity  of  the  anti- 
alexic  property  in  the  antitoxin  is  much  greater. 

To  demonstrate  the  anti-alexic  property  a  mixture  of  antitoxin 
(55  degrees)  and  a  rather  large  dose  of  fresh  hemolytic  serum  is 
used.  For  example,  two  parts  of  antitoxin  and  one  part  of  hemo- 
toxin  are  mixed  together  and  rabbit  corpuscles  added.  These 


200  STUDIES  IN  IMMUNITY. 

corpuscles  remain  intact  even  after  a  long  time.  But  if  a  small 
amount  of  fresh  rabbit  serum  is  added  to  the  mixture,  a  rapid  dis- 
solution takes  place,  although  this  added  serum  is  in  itself  harmless 
for  the  corpuscles.  This  shows  that  the  original  mixture  contained 
an  excess  of  sensitizing  substance,  as  demonstrated  by  the  destruc- 
tion of  the  corpuscles  on  the  addition  of  normal  rabbit  serum. 
Consequently,  although  the  original  mixture  did  not  affect  the  cor- 
puscles, there  was  no  lack  of  sensitizing  substance  and  the  protec- 
tion is  due  to  the  neutralization  of  the  guinea-pig  alexin  also 
present  in  the  fresh  hemolytic  serum  by  the  antitoxin. 

To  show  the  anti-alexic  activity  in  another  way  we  prepare  a  mix- 
ture of  1  c.c.  of  fresh  normal  guinea-pig  serum  and  2  or  3  c.c.  of 
antitoxin  (55  degrees).  As  a  control  a  mixture  1  c.c.  of  fresh  guinea- 
pig  serum  and  2  or  3  c.c.  of  normal  rabbit  serum  (55  degrees)  is 
also  made.  To  each  mixture  is  added  a  strong  enough  dose  of  sen- 
sitizing substance  (heated  hemolytic  serum)  to  resist  neutralization 
by  the  antitoxin;  rabbit  corpuscles  subsequently  added  to  each 
mixture  remain  intact  in  the  first  and  are  destroyed  in  the  second. 
To  make  this  experiment  more  conclusive  a  second  control  may  be 
prepared  containing,  like  the  first,  antitoxin  and  sensitizing  sub- 
stance, but  with  the  normal  guinea-pig  serum  replaced  by  an  equal 
amount  of  normal  rabbit  serum.  This  mixture  destroys  corpuscles, 
which  proves  that  the  dose  of  sensitizing  substance  used  was  larger 
than  could  be  destroyed  by  the  antitoxin;  and  it  proves  also  that 
our  antitoxin  although  effective  against  guinea-pig  alexin  has  no 
effect  on  rabbit  alexin. 

The  idea  that  antitoxin  has  only  a  weak  "  antisensitizing "  power 
whereas  it  has  a  very  much  more  distinct  "  anti-alexic "  power 
explains  very  clearly  the  fact  that  we  have  already  noted,  namely, 
that  heated  antitoxin  combats  the  effect  of  fresh  hemolytic  sera 
better  than  non-heated  antitoxin.  A  moderate  dose  of  antitoxin 
added  to  fresh  hemolytic  serum  may  neutralize  the  alexin  of  this 
hemolytic  serum,  but  leave  a  certain  amount  of  sensitizing  substance 
still  active. 

If  the  antitoxin  has  not  been  heated,  it  contains  an  additional 
dose  of  alexin,  which  aids  in  the  destruction  of  corpuscles  thr  scn- 
sitization  of  which  it  has  not  been  able  to  prevent.  When  heated 
to  55  degrees  it  does  not  have  this  disadvantage;  it  has  then  only 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  201 

one  of  the  active  substances  of  the  hemolytic  serum  to  neutralize 
in  order  to  protect  the  corpuscles,  namely,  the  alexin.  Before 
heating  it  must  neutralize  completely  not  only  the  alexin  but  also 
the  sensitizing  substance. 

It  is  very  probable  that  the  antisensitizing  substance  and  the  anti- 
alexin  are  two  distinct  substances.  An  antitoxin  supersaturated  with 
sensitizing  substance  still  retains  intact  its  property  of  neutralizing 
normal  guinea-pig  alexin. 

Antihemolytic  and  antibactericidal  properties  of  the  antitoxin :  — 
In  bleeding  three  guinea-pigs,  the  first  normal  without  any  treat- 
ment, the  second  treated  by  injection  of  rabbit  blood,  the  third 
immunized  against  cholera  vibrio,  we  know  that  we  shall  obtain 
three  different  sera  each  possessing  the  same  alexin.  The  identity 
of  the  alexin  of  normal  serum  with  that  of  immune  serum  is  one  of 
the  important  points  in  the  theory  we  offered  five  years  ago  to  ex- 
plain the  mode  of  action  of  preventive  sera  on  vibrios,  a  theory  to 
which  we  shall  later  return.  Two  of  these  three  sera  are  immune 
sera,  and,  although  identical  as  regards  alexin,  differ  profoundly  in 
their  antibodies  or  sensitizing  substances.  The  identity  of  the 
alexin  in  normal  and  immune  sera  explains  why  normal  serum  can 
restore  the  original  destructive  properties  of  various  heated  im- 
mune sera  deprived  of  their  alexin  indifferently.  If,  therefore,  we 
add  to  normal  guinea-pig  serum  containing  alexin  an  anti-alexin 
neutralizing  this  latter  substance,  the  normal  serum  should  become 
incapable,  not  only  of  dissolving  sensitized  red  blood  cells,  but  also 
of  producing  a  granular  transformation  in  cholera  vibrios  treated 
with  cholera-sensitizing  substance.  As  our  antitoxin  neutralizes 
guinea-pig  alexin  we  should  expect  it  to  protect  various  sensitized 
cells  against  the  effect  of  this  alexin.  Such  an  antitoxin,  therefore, 
would  be  both  antihemolytic  and  antibactericidal. 

Experiment  confirms  these  suppositions.  On  mixing  antitoxin 
(55  degrees)  with  a  certain  amount  of  fresh  normal  guinea-pig  serum 
we  remove  both  its  property  of  restoring  cellulicidal  activity  to  hemo- 
lytic serum  and  also  the  power  of  restoring  the  bactericidal  property 
to  anticholera  serum  (55  degrees).  But  if  we  use  normal  rabbit  serum 
instead  of  normal  guinea-pig  serum  the  antitoxin  no  longer  shows 
any  protective  property  either  for  vibrios  or  corpuscles.  Although 


202  STUDIES  IN   IMMUNITY. 

active  against  guinea-pig  alexin  it  has  no  effect  on  rabbit  alexin. 
Experimental  details  follow: 

An  emulsion  of  sensitized  cholera  vibrios^,  prepared  by  suspending  an  agar 
culture  in  10  c.c.  of  salt  solution,  and  adding  2  c.c.  of  cholera  serum  from  a  rabbit 
heated  to  56  degrees,  is  used. 

I.  MIXTURES  CONTAINING  SENSITIZED  VIBRIOS. 

a.  Fresh  normal  guinea-pig  serum,  0.2  c.c.;  antitoxin  (55  degrees),  0.4  c.c. 

b.  Fresh  normal  guinea-pig  serum,  0.2  c.c. ;  normal  rabbit  serum  (55  de- 
grees), 0.4  c.c. 

c.  Fresh  normal  rabbit  serum,  0.2  c.c.;  antitoxin  (55  degrees),  0.4  c.c. 

d.  Fresh  normal  rabbit  serum,  0.2  c.c. ;  normal  rabbit  serum  (55  degrees)  ,0.4  c.c. 
To  each  of  these  four  mixtures  0.2  c.c.  of  an  emulsion  of  sensitized  vibrios  is 

then  added.  The  mixtures  are  left  for  an  hour  at  37  degrees.  Granular  trans- 
formation is  complete  in  mixtures  6,  c,  and  d.  The  vibrios  in  mixture  a,  con- 
taining guinea-pig  alexin  neutralized  by  antitoxin,  on  the  contrary,  have  kept 
their  normal  appearance. 

II.  MIXTURES  CONTAINING  SENSITIZED  BLOOD. 

This  blood  consists  of  0.2  of  a  cubic  centimeter  of  rabbit  blood  (previously 
washed  in  salt  solution)  plus  1  c.c.  of  hemolytic  serum  heated  to  55  degrees. 

e.  Identical  with  a. 

f.  Identical  with  6. 

g.  Identical  with  c. 

To  each  of  these  mixtures  0.1  c.c.  of  the  sensitized  blood  is  added.  The  cor- 
puscles are  rapidly  dissolved  in/,  less  rapidly  in  g  and  remain  intact  in  c. 

For  the  sake  of  completeness  we  may  mention  that  the  bacteri- 
cidal effect  is  evident,  not  only  by  granular  transformation  of  the 
vibrios,  but  by  their  complete  destruction,  as  shown  by  inoculating 
agar  tubes  with  the  mixtures.  In  this  way  we  find  that  a  small 
amount  of  sensitized  vibrios  mixed  with  normal  guinea-pig  serum 
plus  normal  rabbit  serum  (55  degrees)  becomes  completely  sterile. 
The  same  dose  of-  vibrios  mixed  with  guinea-pig  serum  plus  anti- 
toxin from  the  rabbit  (55  degrees),  on  the  contrary,  remains  alive; 
cultures  from  this  mixture  on  gelatin  at  intervals  show  numerous 
colonies. 

The  specificity  of  the  anti-alexic  action  of  the  antitoxin.  — Our  anti- 
toxin neutralizes  the  alexin  of  guinea-pig  serum.  We  have  already 
noted  that  it  has  no  effect  on  the  alexin  of  rabbit  serum.  Does  it 
affect  the  alexins  in  sera  from  other  animal  species?  This  may  be 
answered  experimentally  by  adding  doses  of  fresh  serum  from  differ- 
ent animals  to  a  mixture  composed  as  follows : 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  203 

Antitoxin,  55  degrees;  fresh  alexic  serum  to  be  tested;  sensitizer 
in  sufficient  dose  (that  is,  hemolytic  serum  heated  to  55  degrees). 
The  effect  of  this  mixture  on  subsequently  added  rabbit  corpuscles 
will  indicate  whether  or  not  the  alexin  in  the  serum  added  is  neu- 
tralized by  the  antitoxin. 

A  control  series  is  made  with  similar  mixtures  containing  normal 
rabbit  serum  (55  degrees)  in  place  of  antitoxin.  This  series  shows 
the  intensity  of  corpuscle  destruction  by  the  combined  action  of 
the  sensitizer  and  the  normal  sera  when  antitoxin  is  absent. 
A  third  series  containing  normal  rabbit  serum,  55  degrees,  plus  the 
fresh  normal  sera  under  consideration  but  without  sensitizer  is 
also  useful;  this  series  indicates  what  dissolving  properties  are 
present  in  the  normal  sera  alone  without  any  sensitizer.  The 
rabbit  blood  added  to  these  mixtures  should  previously  have  been 
Washed  in  salt  solution  in  order  to  free  the  corpuscles  from  rabbit 
serum. 

Without  going  into  minute  details  of  the  results  of  such  experi- 
ments we  may  state  that  the  antitoxin  neutralizes  guinea-pig  alexin 
efficiently,  but  has  no  effect  on  alexin  from  the  rat,  dog,  rabbit,  goat, 
goose  or  hen. 

It  does,  however,  have  a  distinct  neutralizing  effect  on  pigeon 
alexin.  We  may  therefore  conclude  that  this  anti-alexic  activity 
is  relatively  but  not  absolutely  specific.  With  a  certain  exception 
(pigeon  alexin)  our  anti-alexin  has  no  neutralizing  effect  on  any 
animal  species  tested  except  the  guinea-pig.  These  results  confirm 
distinctly  the  idea  that  the  alexin  in  the  sera  of  different  animal 
species  is  not  uniformly  identical;  this  idea  was  already  probable 
from  the  fact  that  the  corpuscles  of  a  given  species  are  attacked  by 
foreign  normal  sera  with  an  intensity  that  varies  according  to  the 
species  from  which  this  serum  has  been  obtained. 

The  direct  action  of  the  antitoxin  on  the  toxin.  —  Our  anti-alexin 
counteracts  the  harmful  effect  of  guinea-pig  alexin  on  rabbit  cor- 
puscles. There  are  reasons,  moreover,  for  believing  that  anti-alexin 
combines  with  alexin  or  acts  directly  on  this  toxic  substance  by 
destroying  or  modifying  it.  This  conclusion,  however,  cannot  be 
accepted  offhand,  as  proven.  We  might  imagine,  perhaps,  although 
it  seems  scarcely  reasonable,  that  antitoxin  has  no  effect  on  the 


204  STUDIES  IN   IMMUNITY. 

alexin,  but  that  in  some  way  it  prevents  the  corpuscles  from  yielding 
to  its  harmful  effect.  As  is  well  known,  various  observers  and  in 
particular  Cherry  and  Martin,  in  their  study  of  other  toxins  and 
antitoxins,  have  offered  certain  facts  that  favor  the  hypothesis  that 
antitoxins  act  directly  on  toxins. 

It  is  worth  while  considering,  however,  whether  this  hypothesis  is 
exact  so  far  as  our  toxin  and  antitoxin  are  concerned.  Two  ques- 
tions immediately  arise  on  consideration  of  this  subject:  first,  if  to 
the  first  of  two  tubes  containing  the  same  amount  of  antitoxin  there 
is  added  a  little  fresh  alexin  and  to  the  second  the  same  amount  of 
serum  heated  to  55  degrees,  and  if  both  mixtures  are  then  heated 
to  55  degrees,  shall  we  find  that  their  antitoxic  effect  on  a  new  dose 
of  fresh  active  serum  is  the  same?  Or  this  question  may  be  ex- 
pressed in  another  way :  when  alexin  has  been  heated  to  55  degrees, 
and  has  thereby  lost  its  toxicity  for  corpuscles,  will  it  still  neutralize 
as  much  antitoxin  as  it  could  before  being  heated?  Second:  does  a 
neutral  mixture  of  alexin  and  antitoxin  heated  to  55  degrees  become 
antitoxic  (by  a  neutral  mixture  we  mean  a  mixture  that  has  little 
or  no  effect  on  sensitized  corpuscles)? 

If  antitoxin  and  alexin  are  not  combined,  but  continue  to  exist 
side  by  side  in  a  free  condition,  heating  which  destroys  the  toxin 
(alexin),  but  has  no  effect  on  antitoxin,  might  leave  this  latter  sub- 
stance intact.  And  further,  if  the  antitoxin  does  not  act  directly 
on  the  alexin  in  the  two  mixtures  under  question,  we  should  have 
the  same  antitoxic  value  after  heating  to  55  degrees.  As  a  matter 
of  fact  the  activity  or  non-activity  of  the  alexin  added  to  antitoxin 
should  not  affect  the  result.  For  the  sake  of  clearness  we  shall 
answer  these  questions  at  once  and  then  consider  the  experiments 
by  means  of  which  we  arrived  at  these  answers :  first,  alexin  heated 
to  55  degrees  and  deprived  of  its  cellulicidal  activity  has  lost  wholly 
or  to  a  great  extent  its  power  of  saturating  antitoxin;  second,  the 
antitoxin  is  not  recovered  from  a  neutral  mixture  of  antitoxin  and 
fresh  alexin  by  heating  it  to  55  degrees,  although  this  temperature 
destroys  the  activity  of  the  toxin.  The  antitoxin  must  then  have 
been  definitely  neutralized. 

Both  these  results  lead  to  the  conclusion  that  antitoxin  acts 
directly  on  the  toxin  by  destroying  its  toxic  activity.  The  experiment 
to  prove  these  facts  follows: 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  205 

It  was  first  determined  that  two  parts  of  antitoxin  (56  degrees)  will  neutralize 
one  part  of  alexin  (fresh  normal  guinea-pig  serum) .  Two  mixtures  were  prepared 
as  follows: 

a.  Antitoxin,  2  c.c.;  fresh  normal  guinea-pig  serum,  1  c.c. 

b.  Antitoxin,  2  c.c.;  normal  guinea-pig  serum  heated  to  55  degrees,  1  c.c. 
As  a  control  the  following  mixtures  were  prepared: 

c.  Antitoxin,  2  c.c.;  salt  solution,  1  c.c.     In  this  mixture  the  antitoxin  is 
simply  diluted. 

d.  Normal  rabbit  serum  (55  degrees),  2  c.c.;  salt  solution,  1  c.c. 

These  four  mixtures  are  then  heated  for  half  an  hour  to  from  55  to  56  degrees. 
The  mixtures  are  subjected  to  exactly  similar  conditions,  but  mixture  a,  con- 
taining fresh  alexin,  is  naturally  the  one  most  affected,  as  the  alexin  is  destroyed 
at  this  temperature. 

After  heating,  the  antitoxic  value  of  the  four  mixtures  is  determined.  For  this 
purpose  a  suitable  dose  of  fresh  normal  guinea-pig  serum  is  added  to  each  tube.* 

Later  well-sensitized  rabbit  blood  is  added  to  each  tube.  The  amount  of  sensi- 
tizer  used  is  sufficiently  large  to  prevent  the  antisensitizing  effect  of  the  antitoxin 
from  being  noticeable. 

It  is  evident  that  the  blood  will  hemolyze  in  mixtures  in  which  the  toxin  (alexin) 
has  not  been  neutralized ;  in  other  words,  in  mixtures  that  do  not  contain  active 
anti-alexin.  Hemolysis  takes  place  quickly  in  d,  which  contains  no  antitoxin. 
Corpuscles  remain  intact  in  c,  containing  a  mixture  of  antitoxin  and  salt  solu- 
tion. Mixtures  a  and  b  do  not  act  exactly  alike.  In  6  the  corpuscles  remain 
intact,  as  in  c,  for  a  long  time,  but  finally  become  partially  hemolyzed;  in  mixture 
a  hemolysis  is  very  energetic.  The  contrast  between  a  and  b  is  very  striking. 
In  a  there  is  only  a  slight  antitoxic  effect:  unheated  normal  serum  neutralizes 
antitoxin  much  more  distinctly  than  does  heated  serum. 

We  shall  not  consider  farther  for  the  present  the  mechanism  by 
which  the  antitoxic  serum  protects  corpuscles  from  the  hemotoxin, 
but  shall  hope  to  return  later  to  a  consideration  of  this  question. 
We  may  mention  in  conclusion  that  the  antitoxin  has  also  anti- 
agglutinating  properties  against  the  hemolytic  serum.  It  also 
causes  a  precipitate  both  with  hemolytic  serum  and  also  with  normal 
guinea-pig  serum.  This  precipitating  property  exists,  not  only  in 
animals  that  furnish  antitoxin  (i.e.,  rabbits  treated  with  hemolytic 
serum),  but  also  in  rabbits  given  injections  of  defibrinated  blood  or 
normal  guinea-pig  serum.  We  refer  to  our  previous  article  for  more 
information  on  this  property  of  serum,  f 

*  The  amount  of  normal  serum  that  is  best  to  add  is  determined  by  using  a 
series  of  tubes  corresponding  to  each  mixture  a,  b,  etc.,  to  each  one  of  which  a 
different  amount  of  alexin  is  added. 

t  We  may  recall  simply  that  the  "precipitating  property"  of  such  a  serum  is 
quite  distinct  from  its  antitoxic  property.  Nor  has  it  any  relation  to  the  agglu- 
tinating property  of  the  serum  for  corpuscles. 


206  STUDIES  IN  IMMUNITY. 

III.    OBSERVATIONS    ON   THE   THEORIES   OF   CHEMICAL 
IMMUNITY. 

Immunity  is  due  primarily  to  phagocytic  activity,  that  indis- 
pensable function  of  the  body  in  the  taking  up  and  destruction  of 
bacteria  or  alien  cells.  Substances  having  a  digestive  effect  on  bac- 
teria may  also  be  found  in  the  blood  serum,  particularly  during  the 
condition  of  artificial  immunity.  As  our  knowledge  of  these  active 
substances  of  serum  has  increased  it  has  become  evident  that  they, 
too,  owe  their  origin  to  those  phagocytic  cells  the  function  of  which 
is  to  protect  the  animal  body. 

The  cytolytic  properties  of  serum  have  come  to  be  regarded  more 
and  more  as  simply  a  new  manifestation  of  the  activity  of  the  pro- 
tective cells.  The  tendency  in  the  study  of  immunity  has  been  to 
harmonize  these  humoral  manifestations  with  the  functions  of  the 
phagocytes,  which,  as  Metchnikoff  has  shown,  are  both  in  origin 
and  function  the  digestive  cells,  fitted  to  form  substances  that  digest 
and  destroy  alien  cells. 

But  these  substances  in  serum  are  themselves  worthy  of  study 
apart  from  any  consideration  of  their  origin.  The  study  of  this 
"immunity  of  a  chemical  nature "  has  consisted  hitherto  in  a  com- 
parison between  the  sera  of  normal  animals  and  the  sera  of  animals 
that  are  in  a  condition  of  artificial  immunity.  In  the  sera  of  these 
latter  animals,  particularly,  the  existence  of  specific  cellulicidal 
properties,  frequently  of  great  intensity,  has  been  noted.*  But 
this  cytolytic  power  is  not  the  only  characteristic  of  immune  sera; 
they  have  also  the  curious  property  of  creating  on  injection  an 
intense  cellulicidal  property  in  the  serum  of  a  normal  animal,  as 
Fraenkel  and  Sobernheim  first  pointed  out  in  1894.  It  is  notable 
that  immune  sera  still  retain  this  transferring  property,  even  after 
they  have  lost  their  own  power  to  destroy  cells  by  being  heated  to 
55  degrees.  In  the  example  given  by  Fraenkel  and  Sobernheim, 
cholera  serum,  whether  intact  or  heated  to  from  55  degrees  to  60 
degrees,  when  injected  into  a  normal  animal  endows  the  latter's 
serum  with  an  intense  bactericidal  power.  As  another  example  it 
may  be  noted  that  hemolytic  serum  from  a  rabbit  "vaccinated" 

*  We  own  our  conception  of  the  specificity  of  bactericidal  phenomena  to 
Pfeiffer's  researches  particularly. 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  207 

with  hen  blood,  on  injection  into  a  normal  rabbit,  confers  on  the 
serum  of  the  latter  an  intense  hemolytic  power  for  hen  corpuscles.* 
For  the  sake  of  completeness  we  may  add  that  the  serum  of  the 
normal  animal  that  has  received  the  immune  serum  has  a  cellu- 
licidal  property  quite  as  specific  as  that  of  the  immune  serum 
employed.  We  demonstrated  in  1895  that,  when  a  guinea-pig  is 
injected  with  an  immume  serum  specific  for  the  vibrio  Metchnikovi, 
its  serum  acquires  a  bactericidal  property  only  against  the  vibrio 
Metchnikovi  and  not  against  such  vibrios  as  the  V.  choleras. 

How  may  the  destructive  effect  of  a  cytolytic  serum  be  detected 
in  the  affected  cell?  What  lesion  is  produced  when  this  cell  is 
acted  on  by  the  serum  which  indicates  the  destructive  effect?  It 
varies  naturally  with  the  nature  of  the  cell  in  question.  In  vibrios 
it  consists  in  a  granular  transformation;  in  red  blood  cells,  in  hemol- 
ysis.  As  is  well  known,  the  granular  metamorphosis  of  the  cholera 
vibrio  was  first  noted  by  Pfeiffer  in  the  peritoneal  cavity  of  actively 
or  passively  immunized  animals.  This  investigator  thought  that 
this  modification  of  the  vibrio  could  be  brought  about  only  in  the 
animal  body  and  never  in  vitro;  according  to  him  this  transforma- 
tion is  indicative  of  an  essentially  vital  action,  and  is  distinct  from 
the  bactericidal  effect  of  cholera  serum  in  vitro.  According  to  this 
author  there  would  be  two  distinct  varieties  of  bactericidal  action, 
the  one  occurring  exclusively  in  vivo,  and  indicated  by  a  granular 
transformation,  and  the  other  produced  in  vitro,  similar  in  effect 
but  less  marked  and  of  less  significance.  If  this  conception  had 
proved  correct  the  subject  would  have  been  excessively  complicated. 
We  know  now,  however,  that  it  does  not  agree  with  the  facts: 
Metchnikoff,  in  a  classical  experiment  that  inaugurated  the  more 
comprehensive  study  of  these  phenomena,  was  able  to  obtain  the 
granular  transformation  of  vibrios  in  vitro  by  mixing  the  organisms 
with  cholera  serum  plus  the  peritoneal  exudate  from  a  normal 
guinea-pig.  We  subsequently  demonstrated  that  fresh  cholera 
serum  alone  is  able  to  produce  this  metamorphosis,  even  when  quite 
limpid  and  free  from  cells. 

The  important  point  to  be  emphasized  is  that  the  bactericidal 
action  of  a  fluid  or  serum  on  the  cholera  vibrio  is  evidenced  both  in 
vivo  and  in  vitro  by  a  granular  transformation  of  the  organism.  The 

•"•  See  p.  170. 


208  STUDIES  IN   IMMUNITY. 

analogous  action  of  hemolytic  sera  is  detectable  in  vitro  as  well  as 
intraperitoneally  by  a  destruction  of  the  corpuscles  with  a  diffusion 
of  hemoglobin. 

To  summarize :  First,  the  sera  under  consideration  obtained  from 
immunized  animals  are  cytolytic,  the  cytolysis  consisting  in  such 
changes  as  the  granular  transformation  of  vibrios,  and  the  hemolysis 
of  red  blood  cells.  Certain  fluids  from  the  immunized  animals,  as 
the  peritoneal  fluid,  show  similar  cytolytic  effects  in  vivo  or  in  vitro. 
And  second,  a  given  immune  serum  on  injection  into  normal  animals 
confers  on  their  serum  and  body  fluids  similar  cytolytic  properties 
likewise  demonstrable  in  vivo  and  in  vitro.  Immune  serum  trans- 
mits this  property  even  when  itself  deprived  of  its  destructive  prop- 
erty by  heating  to  55  to  56  degrees. 

These  are  the  facts  to  be  coordinated  and  explained  by  a  theory. 
The  first  theory  proposed  was  our  own,  offered  first  in  1895,  and 
confirmed  without  any  essential  addition  by  our  subsequent  re- 
searches. It  might  seem  unnecessary  to  rehearse  our  theory,  as  we 
have  already  outlined  and  repeated  it  several  times.*  This  theory, 
however,  has  not  been  universally  accepted  and  certain  other 
observers  have  preferred  a  different  one:  it  would  therefore  seem 
advisable  to  reconsider  it  and  to  compare  it  with  other  theories. 

Borders  theory  (1895).  — Our  conception  as  expressed  five  years 
ago  is  based  on  the  possibility  (first  demonstrated  by  Metchnikoff) 
of  producing  a  granular  transformation  of  vibrios  in  vitro,  and  de- 
pends particularly  on  the  following  facts: 

1.  Fresh  cholera  serum  produces  a  granular  transformation  of 
cholera  vibrios ;  the  phenomenon  produced  in  vitro  is  identical  with 
the  one  first  observed  in  the  peritoneal  cavity  by  Pfeiffer;  it  is  just 
as  highly  specific  and  may  be  similarly  employed  for  the  diagnosis 
of  vibrios.    Cholera  serum  has  also  the  power  of  clumping  a  culture 
of  vibrios. f 

2.  When  heated  to  55  degrees  cholera  serum  loses  its  bacterici- 
dal property  and  the  power  of  producing  granular  transformation, 
but  still  retains  its  agglutinating  power. 

*  See  articles  on  pp.  8,  56,  81,  and  134. 

t  This  is  the  first  instance  described  of  the  rapid  agglutination  of  a  micro- 
organism by  a  specific  serum  in  high  dilution. 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  209 

3.  Heated  cholera  serum  recovers  its  original  bactericidal  energy 
on  tke  addition  of  a  normal  serum  that  is  itself  only  faintly  bac- 
tericidal.   A  small  amount  of  intact  or  of  heated  cholera  serum 
suffices  to  confer  an  intense  specific  bactericidal  power  on  a  con- 
siderable amount  of  normal  serum.    This  power  is  evidenced  by  a 
granular  transformation  in  the  added  vibrios.* 

4.  Normal  serum  alone  frequently  causes  a  granular  transfor- 
mation of  vibrios  to  a  less  degree,  particularly  if  the  vibrios  are 
attenuated. 

5.  Normal  serum  heated  to  55  degrees  loses  the  property  of 
restoring  the  bactericidal  activity  to  heated  cholera  serum. 

Our  theory  founded  on  these  facts  is  comprised  in  the  two  follow- 
ing propositions: 

A.  The  bactericidal  property  of  cholera  serum,  or  of  analogous 
sera,  is  due  to  the  presence  of  two  distinct  substances:  the  first, 
which  may  be  called  preventive  substance  or  antibody  (later  called 
sensitizer),  is  characteristic  of  immune  serum,  is  specific  and  resists 
a  temperature  of  55  to  60  degrees  and  even  more.  The  other,  or 
proper  bactericidal  substance,  the  alexin,  occurs  in  the  serum  of 
normal  as  well  as  of  vaccinated  animals,  and  is  destroyed  on  heating 
to  55  degrees.  Heating  cholera  serum  to  55  degrees  does  not 
destroy  the  preventive  substance,  but  simply  eliminates  the  alexin. 
As  this  alexin  is  present  in  normal  serum  the  addition  of  such  serum 
restores  to  heated  cholera  serum  its  original  activity.! 

In  such  a  mixture  cholera  serum  again  contains  both  substances 
originally  present,  the  collaboration  of  which  is  essential  in  pro- 
ducing intense  specific  bacteriolysis. 

The  preventive  substance,  in  other  words,  heated  cholera  serum, 
is  not  at  all  bactericidal.  Normal  serum  on  account  of  its  alexin 

*  We  advocated  such  a  mixture  in  vitro  as  a  practical  diagnostic  method  for  the 
cholera  vibrio. 

t  We  have  been  surprised  to  find  that  certain  authors,  particularly  in  Germany, 
give  a  very  inexact  historical  account  of  these  ideas,  frequently  attributing  the 
first  consideration  of  these  facts  to  authors  who  have  only  recently  considered  the 
question.  Consequently  we  may  be  permitted  to  refer  to  certain  passages  in  our 
memoir  of  1895.  See,  for  example,  on  p.  58,  and  on  p.  59. 

We  shall  later  quote  other  references  in  the  text,  particularly  when  we  come 
to  consider  the  unity  of  the  bactericidal  substance  in  different  immune  sera, 
and  the  occurrence  of  bactericidal  power  in  the  fluids  of  passively  immunized 
animals. 


210  STUDIES  IN  IMMUNITY. 

has  a  slight  and  non-specific  bactericidal  activity  affecting,  generally, 
only  attenuated  bacteria.  But  in  presence  of  the  preventive  sub- 
stance of  an  immune  serum  the  alexin  energetically  attacks  that 
particular  race  of  bacteria  for  which  the  immune  serum  is  specific. 
The  activity  of  the  alexin  against  other  bacteria  is  not,  however, 
increased. 

In  short,  the  preventive  substance,  in  itself  not  bactericidal,  acts 
specifically  on  a  cell  by  increasing  the  destructive  effect  of  the 
alexin  on  it.  We  now  call  the  preventive  substance  the  "  sensitizer  " 
as  it  sensitizes  the  cell  to  the  alexin. 

It  may  be  stated  at  this  point  that  the  specific  hemolytic  sera, 
first  demonstrated  by  us  in  1898,  agree  in  general  characteristics 
with  the  bacteriolytic  sera,  and  any  conception  of  one  generally 
applies  to  the  other.  The  majority  of  observers  and  particularly 
Ehrlich  and  Morgenroth  agree  with  us  on  this  point.  The  only 
difference  lies  in  the  cell  affected,  and  hemolysis  corresponds  to 
bacteriolysis. 

B.  When  an  immune  serum,  as,  for  example,  cholera  serum,  is 
given  to  a  normal  animal,  the  introduction  of  the  preventive  sub- 
stance (sensitizer)  transmits  the  specific  bactericidal  power  to  this 
animal's  body  fluids.  The  introduction  of  an  alexin,  present,  of 
course,  in  fresh  immune  serum,  is  not  necessary  and  therefore  a 
heated  immune  serum  will  serve  equally  well.  As  we  stated  in  1895, 
"It  is  quite  evident  why  the  presence  of  the  bactericidal  sub- 
stance (alexin)  is  not  indispensable  for  the  preventive  activity  of 
serum.  It  is  not  characteristic  of  this  serum  alone,  but  is  present 
in  normal  serum  as  well,  and  the  animal  injected  with  cholera  serum 
already  possesses  this  substance,  so  that  its  addition  is  unneces- 
sary. What  the  normal  animal  does  not  possess  is  the  preventive 
substance,  and  it  is  this  substance,  then,  that  must  be  given."  As 
soon  as  the  animal  obtains  the  preventive  substance  it  has  the  two 
factors  necessary  for  intense  specific  bacteriolysis.  On  bleeding 
such  an  animal  we  find  that  its  serum  contains  the  preventive  sub- 
stance as  well  as  the  normally  present  alexin  and  will,  therefore, 
destroy  the  vibrio.  "The  encounter  of  the  two  substances  pro- 
duces as  energetic  a  bactericidal  power  in  the  animal  body  as  in  a 
test  tube." 

As  we  have  already  stated,  this  theory  is  also  applicable  to  hemo- 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  211 

lytic  sera.*  The  important  fundamental  idea  is  that  the  bacterici- 
dal (or  cellulicidal)  substance  in  various  immune  sera  that  endows 
them  with  their  properties  is  similar  to  the  one  found  in  normal 
serum.  This  is  the  principle  of  the  unity  of  the  cytolytic  substance 
which  we  expressed  in  1895  by  saying  that,  in  normal  and  in  vacci- 
nated animals,  "the  bactericidal  substance  is  in  general  respects  the 
same,  whatever  may  be  the  invading  organism.  In  animals  vacci- 
nated against  a  given  infective  agent  the  bactericidal  energy  affects 
that  bacterium  particularly,  owing  to  the  presence  of  the  specific 
preventive  substance,  which  varies  according  to  the  micro-organ- 
ism used  in  immunizing.  It  is  owing  to  the  intervention  of  this  pecul- 
iar preventive  substance  that  the  animal  directs  its  destructive  energy 
against  a  given  infective  agent." 

In  this  description  the  word  "bacterium"  may  be  replaced  by 
the  word  "cell,"  referring  to  bacterium  or  blood  corpuscles,  as  the 
case  may  be.  And  further,  a  reservation  should  be  made,  based  on 
the  subsequent  study  of  hemolysis,  that,  although  the  alexin  is  the 
same  in  normal  and  vaccinated  animals  of  the  same  species,  there 
are  certain  differences  in  the  alexins  from  different  animal  species. 
The  alexins  of  most  animals,  however,  have  the  same  essential  char- 
acteristics. We  have  already  made  another  reservation,  namely, 
that  certain  normal  sera  contain,  beside  the  alexin,  other  bactericidal 
substances  of  less  general  import.  The  bactericidal  substance  for 
B.  anthracis  in  rat  serum,  for  example,  is  certainly  not  an  alexin.  f 

As  far  as  the  absolute  identity  of  the  hemolytic  with  the  bacterio- 
lytic  alexin  in  a  given  serum  is  concerned,  it  would  seem  to  have 
been  firmly  established  in  the  present  article. 

The  fact,  moreover,  that  "it  is  owing  to  the  intervention  of  the 
sensitizer  that  the  animal  directs  its  particular  cellulicidal  activity 
against  a  given  cell"  is  fully  confirmed  by  the  experiments  detailed 
in  the  first  part  of  this  article,  experiments  that  prove,  not  only  that 
the  same  normal  serum,  but  that  the  same  alexin  destroys  at  one  time 
a  vibrio  and  at  another  a  red  blood  cell  depending  on  whether  a 
hemosensitizer  or  a  cholera  sensitizer  is  used. 

k*  It  is  probably  also  applicable  to  the  immune  sera  active  against  other  cells, 
such  as  the  spermotoxic  serum  discovered  by  Landsteiner,  the  leucotoxic  serum 
)f  Metchnikoff,  and  the  epitheliotoxic  serum  of  V.  Dungern. 
t  Rat  serum  has  in  addition  to  this  substance  an  alexin  similar  to  that  of  other 
jera.     See  note  on  p.  180. 


212  STUDIES   IN  IMMUNITY. 

Our  theory  makes  two  assumptions:  First,  that  cytolytic  phe- 
nomena in  vitro,  produced  by  adding  the  sensitive  cell  to  the  two 
substances  of  active  serum,  are  quite  similar  to  those  which  occur  in 
vivo  with  the  same  substances.  This  may  indeed  be  experiment- 
ally demonstrated  provided  that  relatively  similar  amounts  of  each 
substance  are  present  in  both  cases.  For  example,  we  find  that  the 
smallest  amount  of  cholera  sensitizer  that  will  cause  a  given  amount 
of  vibrios  to  be  transformed  by  alexin  in  vitro  is  similar  to  the 
amount  necessary  to  destroy  the  same  number  of  vibrios  in  a  nor- 
mal peritoneal  cavity.  This  fact  has  been  already  mentioned. 

Second,  that  a  normal  animal  injected  with  an  immune  serum 
acts  simply  as  a  passive  container  for  the  sensitizer.  The  animal 
receives  the  substance,  but  does  not  modify  it,  and  it  becomes  diluted 
in  the  fluids  of  the  body,  particularly  in  the  blood.  The  serum 
from  this  injected  animal  should  act,  then,  like  a  dilution  of  the  sen- 
sitizer in  normal  serum.  This  we  believe  we  have  proved  experi- 
mentally.* This  conclusion  holds  as  well  for  the  agglutinating 

property.f 

*** 

Pfeiffer1  s  theory,  (1896). — The  preceding  theory  has  not  been 
the  only  one  to  awaken  interest  in  the  study  of  bactericidal  sera. 
A  year  later  Pfeiffer  offered  an  explanation  of  the  granular  trans- 
formation of  vibrios  that  differs  from  our  own  in  several  important 
particulars.  Pfeiffer,  who  has  carefully  studied  the  bacteriolytic 
changes  in  vibrios  in  the  peritoneal  cavity,  draws  a  sharp  dis- 
tinction between  such  phenomena  and  those  that  take  place  in 
vitro;  the  latter,  according  to  this  investigator,  are  much  less 
energetic.  As  Pfeiffer  understands  it,  vaccinated  animals  have  a 
specific  antibody  that  has  two  forms :  a  stable  and  inactive  form 
that  may  be  kept  for  a  long  time,  and  an  active  bactericidal  form 
which  is  less  stable.  The  antibody  can  easily  change  from  one 
form  to  another.  The  change  from  inactive  to  active  form  is 
brought  about  by  means  of  a  ferment-like  substance  of  cellular 
origin;  this  change  takes  place  easily  in  the  animal  body,  so  that 
the  antibody  can  attack  the  vibrios  in  its  most  destructive  form. 
Serum,  on  the  contrary,  contains  the  antibody  in  its  stable,  inac- 

*  See  pp.  167  to  171. 

f  See,  "The  mode  of  action  of  preventive  sera,"  p.  81. 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  213 

tive  form,  as  it  is  stored  up  against  possible  necessity  in  the  body. 
In  this  latter  form  the  antibody  is  incapable  of  destroying  vibrios. 
To  be  sure,  Pfeiffer  does  not  deny  that  a  granular  transformation 
may  occur  in  vitro  with  fresh  cholera  serum,  but  he  thinks  this 
effect  is  due  to  a  small  amount  of  the  ferment  substance  in  the 
serum  that  renders  the  antibody  bactericidal.  It  is  evident  that 
Pfeiffer  has  been  obliged  to  agree  essentially  with  our  point  of 
view  in  order  to  explain  bacteriolysis  in  vitro,  at  least  to  the  extent 
of  admitting  that  two  substances  are  necessary.  The  resemblance 
between  the  two  theories,  however,  is  only  superficial. 

According  to  Pfeiffer  and  his  pupils  the  substance  present  in 
fresh  normal  serum,  that  has  been  named  alexin,  would  not  be  in  the 
strict  sense  a  bactericidal  substance,  but  rather  the  ferment  which 
transforms  the  inactive  antibody  into  its  active  form.  The  trans- 
formation of  vibrios  in  vitro  by  fresh  cholera  serum  would  be  due  to 
the  effect  of  the  active  antibody  and  not  to  a  direct  action  of  the 
alexin,  the  one  function  of  which  is  to  produce  the  active  antibody. 
Consequently  the  real  cellulicidal  substance  would  always  be  specific ; 
instead  of  being  the  same  in  various  immune  sera,  as  we  think  of 
it,  it  would  vary  in  each  one,  since  the  antibodies  in  immune  sera 
are  essentially  different. 

But  if  the  alexin  bears  no  direct  causal  relation  to  alterations 
shown  by  vibrios  and  corpuscles,  why  do  normal  sera,  which  have 
no  specific  antibodies,  show  a  distinct  if  inferior  bactericidal  effect 
on  vibrios?  How,  as  Buchner  has  emphasized  in  his  valuable 
researches,*  can  these  normal  sera  produce  a  certain  amount  of 
hemolysis? 

This  hemolysis,  by  normal  serum,  although  less,  is  nevertheless 
quite  comparable  to  that  produced  by  hemolytic  sera.  Artificial 
immunization,  as  indicated  by  the  production  of  specific  antibodies, 
does  not  produce  cytolytic  power,  but  renders  it  specifically  more 
intense  by  forming  a  sensitizer  or  specific  antibody.  All  this  tends 
to  invalidate  Pfeiffer's  theory. 

This  theory,  to  be  sure,  would  seem  more  reasonable  if  cytolytic 
phenomena  in  vitro  were  much  less  intense  than  those  in  the  animal 
body  (peritoneal  cavity).  There  is,  however,  no  difference  in  inten- 

*  Buchner,  as  we  know,  brought  out  the  fundamental  fact  that  normal  serum 
heated  to  55  degrees  loses  both  its  bactericidal  and  globulicidal  properties. 


214  STUDIES  IN  IMMUNITY. 

sity,  as  we  have  already  stated.  To  be  sure,  if  a  mixture  is  made  in 
vitro  containing  too  little  cholera  serum  in  proportion  to  the  vibrios 
to  give  complete  bacteriolysis,  the  injection  of  this  mixture  into  the 
peritoneal  cavity  of  a  normal  animal  will  frequently  result  in  a 
granular  transformation.  It  is  evident,  however,  that  by  this  pro- 
cedure we  increase  the  amount  of  alexin  (peritoneal  exudate),  and 
such  an  increase  of  one  of  the  two  substances  required  for  bacteri- 
olysis naturally  produces  a  greater  effect.*  To  estimate  bactericidal 
power  correctly  either  in  vitro  or  in  vivo  the  vibrios  used  must 
be  mixed  with  comparable  amounts  of  the  two  active  substances; 
if  this  precaution  is  observed  it  will  be  found  that  vibrios  or  cor- 
puscles are  altered  as  well  in  vitro  as  in  vivo. 

The  properties  of  the  antitoxin  studied  in  the  preceding  pages 
suggests  another  objection  to  Pfeiffer's  theory. 

Let  us  mix  a  certain  amount  (e.g.,  0.4  of  a  cubic  centimeter)  of 
hemolytic  serum,  55  degrees  (sensitizer),  with  a  little  fresh,  normal 
guinea-pig  serum  (0.2  of  a  cubic  centimeter).  Similar  tubes  are 
prepared  containing,  in  the  place  of  guinea-pig  alexm,  rabbit  and 
rat  alexin  respectively.  Any  one  of  these  mixtures  is  strongly 
hemolytic  for  rabbit  blood,  although  no  one  of  the  constituents 
alone  is  so.  What  has  happened  according  to  Pfeiffer's  theory? 
The  alexin  employed  has  transformed  the  inactive  thermostable 
antibody  into  the  active  and  specifically  globulicidal  antibody. 
And  consequently,  in  such  a  mixture,  it  is  this  active  antibody  and . 
not  the  alexin  that  destroys  the  corpuscles.  It  may  be  added  that 
of  the  three  alexins  mentioned  the  one  from  the  guinea-pig  forms 
the  best  hemolytic  mixture  with  the  sensitizer. 

Since  each  mixture  contains  the  same  hemolytic  substance,  we 
should  expect,  according  to  Pfeiffer's  theory,  that  an  antitoxin 
capable  of  neutralizing  the  destructive  effect  of  one  would  also 

*  We  may  recall  the  point  demonstrated  by  us  in  1895,  that  a  fresh  immune 
serum  does  not  contain  appreciably  more  alexin  than  normal  serum.  The  sen- 
sitizing property  of  an  immune  serum,  however,  is  so  great  that  it  can  sensitize 
many  more  vibrios  than  its  alexin  is  able  to  destroy.  Conversely,  the  amount  of 
alexin  in  a  given  dose  of  immune  serum  is  too  small  to  destroy  all  the  vibrios  that 
the  serum  can  sensitize.  For  this  reason  fresh  immune  serum  is  able  to  destroy 
many  more  vibrios  when  fresh  normal  serum  is  added  on  account  of  the  addi- 
tional amount  of  the  alexin.  On  summing  up  the  facts  on  which  our  theory  is 
based  we  noted  that  a  small  dose  of  immune  seorum  suffices  to  endow  a  relatively 
large  dose  of  normal  serum  with  intense  bactericidal  energy. 


HEMOLYTIC  SERA  AND  THEIR  ANTITOXINS.  215 

neutralize  either  of  the  other  mixtures.  Let  us  add,  then,  to  the 
most  strongly  hemolytic  mixture,  namely  sensitizer  plus  guinea-pig 
alexin,  some  of  our  heated  antitoxin  (0.8  of  a  cubic  centimeter). 
The  mixture  becomes  harmless  for  rabbit  corpuscles.  The  addition 
of  the  same  amount  of  antitoxin  to  either  of  the  other  mixtures  con- 
taining rabbit  or  rat  alexin  does  not  produce  any  neutralizing  effect. 
This  result,  then,  is  contrary  to  a  logical  deduction  from  Pfeiffer's 
theory.*  On  the  contrary,  the  results  are  quite  explicable:  the 
different  alexins  act  directly  on  the  sensitized  corpuscles  to  destroy 
them.  And  the  antitoxin,  being  specific  for  guinea-pig  alexin,  nat- 
urally affects  the  activity  of  this  alexin  only. 

With  this  we  conclude  our  remarks  on  the  theories  of  the  cytolytic 
sera.  To  sum  up,  our  theory,  offered  in  1895,  explains  all  the  facts 
observed  and  is  in  disagreement  with  none  of  them.  From  a  general 
standpoint  this  theory  and  facts  on  which  it  is  founded  contains 
three  general  ideas : 

1.  Artificial  immunization  simply  renders  the  cytolytic  mani- 
festations of  normal  serum  specifically  more  intense.     This  con- 
ception we  demonstrated  by  showing  that  both  normal  and  immune 
serum  (cholera  serum)  produce  a  similar  effect  on  the  cholera  vibrio, 
which  effect,  however,  is  of  unequal  intensity  in  the  two  instances. 
This  conception  has  been  confirmed  by  our  being  able  to  exalt  the 
hemolytic  activity  of  a  given  normal  animal's  serum  by  vaccinating 
the  animal  against  red  blood  cells. 

2.  The  immunized  animal  does  not  have  its  cytolytic  substance 
increased  over  normal,  but  simply  produces  a  large  amount  of  the 
specific  substance  which  favors  the  activity  of  the  cytolytic  sub- 
stance, f 

3.  A  reaction  of  immunity,  consisting  in  the  production  of  an 
antibody,  noted  in  an  animal  injected  with  harmless  cells,  such  as 
red  blood  corpuscles,  is  identical  with  the  reaction  shown  by  an 
animal  to  an  infective  agent,  for  purposes  of  protection. 

*  Such  an  experiment  naturally  comprises  suitable  controls  containing  heated 
normal  rabbit  serum  instead  of  the  antitoxin.  Hemolysis  occurs  in  all  the  control 
mixtures  and  most  markedly  in  the  one  containing  guinea-pig  alexin. 

t  The  mechanism  by  means  of  which  the  sensitizing  substance  favors  activity 
has  recently  given  rise  to  interesting  studies.  Among  them  may  be  mentioned, 
in  particular,  the  researches  of  Ehrlich  and  Morgenroth,  which  have  been  discussed 
in  the  present  article. 


216  STUDIES  IN  IMMUNITY. 

CONCLUSIONS. 

Under  this  heading  we  shall  note  only  the  facts  subjected  to 
experimental  proof  in  the  first  two  sections  of  this  article : 

1.  Corpuscles  sensitized  by  a  given  sensitizer  are  destructible 
by  the  different  alexins  (normal  sera)  of  many  if  not  of  all  animal 
species. 

2.  In  a  given  serum  the  bacteriolytic  is  identical  with  the  hemo- 
lytic  alexin. 

3.  The  fixing  properties  of  corpuscles  for  the  active  substances 
of  hemolytic  sera  lie  in  their  stromata.    This  fixation  is  similar  to 
a  process  of  dyeing. 

4.  An  antitoxin  to  a  hemolytic  serum  may  be  produced.    This 
antitoxin  has  both  an  antisensitizing  and  an  anti-alexic  property. 

5.  Owing  to  its  anti-alexic  property  the  antitoxin  is  both  anti- 
hemolytic  and  antibactericidal. 

6.  The  anti-alexin  would  seem  to  neutralize  the  alexin  by  acting 
directly  upon  it. 

7.  The  anti-alexin  in  question  is  specific,  although  not  absolutely 
so.    It  neutralizes  guinea-pig  alexin,  but  has  no  effect  on  alexins 
from  most  other  animals. 


X.  ON  THE   EXISTENCE  OF  SENSITIZING  SUB- 
STANCES  IN  THE  MAJORITY  OF 
ANTIMICROBIAL  SERA  * 

BY  DRS.  JULES  BORDET  AND  OCTAVE  GENGOU. 

(From  Professor  Metchnikoff's  Laboratory.) 

The  serum  of  many  animals  contains  alexin,  that  ill-defined, 
chemically  unknown  substance  to  which  is  due  the  property  of 
sera  in  general  of  producing  a  harmful  effect  on  various  cells  and  on 
certain  bacteria.  The  activity  of  the  alexin  is  destroyed  on  heating 
serum  to  55  degrees.  Alexin  is  found,  in  relatively  similar  amounts, 
in  the  serum  of  normal  and  of  vaccinated  animals1  artificial  im- 
munization changes  neither  its  amount  nor  its  properties. 

When  an  animal  is  vaccinated  against  the  cholera  vibrio  there  is 
formed  in  the  body  a  particular  substance,  the  preventive  or  sen- 
sitizing substance,  that  resists  heating  to  relatively  high  tempera- 
tures. This  substance  is  not  in  itself  in  any  way  destructive  for 
the  vibrio.  It  does,  however,  faciliate  in  a  specific  manner  the 
destructive  action  of  the  alexin  on  this  micro-organism.  It  may 
further  be  said  that  the  specific  bactericidal  property  of  cholera 
serum,  although  primarily  due  to  the  alexin,  properly  speaking, 
depends  on  the  collaboration  of  two  substances,  the  alexin  and  the 
favoring  or  sensitizing  substance.  This  conception,  offered  by  one 
of  us  in  1895,  explains  the  various  properties  of  cholera  serum.  It 
is  closely  applicable  also  to  the  specific  hemolytic  sera  between 
which  and  cholera  serum  most  evident  analogies  exist.  In  brief, 
the  intense  destructive  power  in  bacteriolytic  or  cytolytic  serum  is 
due  to  the  presence  of  a  specific  antibody,  the  sensitizer,  in  addi- 
tion to  the  ordinary  alexin. 

In  the  preceding  description,  in  referring  to  sera  specific  for  bac- 
teria, we  have  mentioned  cholera  serum  only.  As  a  matter  of  fact 

*  Sur  ^existence  de  substances  sensibilisatrices  dans  la  plupart  des  scrums 
antimicrobiens.  Annales  de  Tlnstitut  Pasteur,  XV,  1901,  290. 

217 


218  STUDIES  IN  IMMUNITY 

it  would  have  been  hasty  to  assert  that  specific  sensitizers  are  present 
in  antimicrobial  sera  in  general.  Hitherto  these  bodies  have  been 
demonstrated  with  certainty  only  in  the  specific  antisera  for  the 
true  cholera  or  other  similar  vibrios. 

This  is  quite  understandable  when  we  come  to  consider  the  known 
method  of  demonstrating  the  sensitizing  substance.  This  method 
described  by  one  of  us  in  the  case  of  cholera  serum,  and  later  applied 
to  hemolytic  sera,  is  as  follows: 

Mixtures  are  made  in  suitable  proportions  of  cholera  vibrios 
with  normal  serum  and  specific  serum  respectively.  In  the  second 
mixture  an  intense  destruction  of  the  bacteria  occurs,  as  is  evidenced 
by  their  complete  granular  transformation.  In  the  first  mixture, 
on  the  contrary,  a  morphologically  similar,  but  relatively  insignifi- 
cant, change  takes  place.  Both  cholera  serum  and  normal  serum 
lose  their  bactericidal  power  completely  on  being  heated  to  55  de- 
grees. But  the  addition  of  a  trace  of  heated  cholera  serum  to 
unheated  normal  serum  forms  a  mixture  that  is  as  strongly  bac- 
tericidal as  fresh  cholera  serum;  it  enables  it  to  produce  granules 
in  the  vibrios.  This  proves  that  heated  cholera  serum,  although 
harmless  alone,  still  favors  the  bactericidal  energy  of  the  alexin  of 
normal  serum. 

This  method,  then,  of  demonstrating  a  sensitizer  depends  on  the 
presence  of  some  microscopically  detectable  lesion  of  the  bacterium 
affected;  bacteriolysis  must  occur.  With  hemolytic  sera  the  cri- 
terion is  the  occurrence  of  hemolysis. 

Not  all  bacteria,  however,  fulfill  this  condition.  Many  of  them 
not  only  are  undestroyed,  but  remain  apparently  unchanged  in 
the  presence  of  serum  from  highly  immunized  animals.  In  such 
cases  the  method  described  is  of  no  avail  and  should  be  replaced  by 
another. 

We  have,  indeed,  another  method  to  offer  for  the  demonstration 
of  sensitizers  in  the  sera  of  animals  immunized  against  such  bacteria 
as  B.  pestis,  first  anthrax  vaccine,  B.  typhosus,  the  bacillus  of 
swine  plague,  and  B.  proteus  vulgaris. 

But  first  we  must  recall  an  experiment  described  a  year  ago  in 
the  Pasteur  Annals  *  the  essentials  of  which  follow : 

If  well-sensitized  rabbit  blood  corpuscles  (that  is,  corpuscles 

*  See  p.  186. 


THE  EXISTENCE  OF  SENSITIZING  SUBSTANCES.  219 

treated  with  a  hemolytic  serum  heated  to  55  degrees)  are  added  to 
fresh  guinea-pig  serum  containing  alexin,  their  dissolution  follows. 
If  after  a  certain  time  sensitized  cholera  vibrios  (i.e.,  vibrios  treated 
with  heated  cholera  serum)  are  added  and  the  mixture  placed  in  the 
incubator,  no  transformation  or  change  takes  place  in  the  vibrios. 
From  this  result  we  know  that  there  was  no  free  alexin  in  the  mix- 
ture, for,  if  present,  it  would  have  transformed  the  vibrios.  From  a 
control  tube  we  learn  that  a  transformation  occurs  in  the  vibrios  if 
the  red  blood  corpuscles  added  in  the  first  place  are  not  sensitized. 

The  converse  of  this  experiment  also  holds.  If  sensitized  cholera 
vibrios  are  added  to  normal  alexic  serum,  subsequently  added  sen- 
sitized corpuscles  are  not  hemolyzed. 

From  such  experiments  we  drew,  it  will  be  recalled,  two  distinct 
conclusions :  First,  corpuscles  or  bacteria  when  sensitized  are  able  to 
absorb  alexin  with  avidity  and  to  remove  it  from  the  surrounding 
fluid;  second,  in  a  given  serum  the  same  alexin  may  produce  either 
hemolysis  or  bacteriolysis.* 

These  conclusions  find  still  further  confirmation  in  the  following 
article.  In  the  present  article  a  fact  suggested  by  the  experiment 
just  outlined  is  of  preeminent  interest:  To  demonstrate  the  existence 
of  a  sensitizer  in  an  antimicrobial  serum  we  may  make  use  of  its 
property  of  causing  the  bacterium  it  affects  to  absorb  alexin. 

As  an  experiment  to  demonstrate  this  fact  is  practically  the  same 
with  any  one  of  the  antimicrobial  sera  studied,  it  may  be  described 
in  detail  once  for  all.  We  shall  take  as  an  example  antiplague 
serum. 

Serum  of  a  horse  vaccinated  against  B.  pestis.  —  This  strongly  pre- 
ventive serum  was  kindly  furnished  us  by  Dr.  Dujardin-Beaumetz, 
who  has  charge  of  preparing  it  and  testing  its  potency  at  the  Pasteur 
Institute. 

This  serum  and  normal  horse  serum  were  heated  to  56°  C  for  one 
half  hour  to  destroy  the  alexin.  A  24-hour  agar  culture  of  B.  pestis 
was  suspended  in  a  small  amount  of  salt  solution  so  as  to  form  a 
thick  emulsion  of  bacteria.  Fresh  guinea-pig  serum  obtained 
by  bleeding  the  guinea-pig  the  day  before,  and  freed  from  cor- 
puscles by  centrifugalization,  was  also  at  hand  and  was  used  for 
alexin. 

*  The  unity  of  the  alexin  in  a  given  serum  is  also  admitted  by  Buchner. 


220  STUDIES  IN  IMMUNITY. 

The  following  mixtures  were  then  prepared  in  test  tubes : 

(a)  Alexic  serum,  0.2  c.c.;  emulsion  of  plague  bacilli,  0.4  c.c.; 
antiplague  serum,  56  degrees,  1.2  c.c. 

(b)  Same  as  "a,"  with  normal  horse  serum,  56  degrees,  replacing 
the  antiplague  serum. 

(c)  Same  as  ua,"  but  containing  no  emulsion  of  plague  bacilli. 

(d)  Same  as  "b,"  but  containing  no  emulsion  of  plague  bacilli. 
Each  of  these  four  mixtures  contains  the  same  amount  of  alexin 

(normal  guinea-pig  serum). 

(e)  Emulsion  of  plague  bacilli,  0.4  c.c.;  antiplague  serum,  56  de- 
grees, 1.2  c.c. 

(f)  Emulsion  of    plague  bacilli,  0.4  c.c.;    normal  horse  serum, 
56  degrees,  1.2  c.c. 

These  two  tubes  are  the  same  as  "a"  and  "b "respectively,  with- 
out alexin. 

The  mixtures  are  left  at  room  temperature  (15  to  20  degrees)  for 
about  5  hours.  To  each  tube  in  turn  is  added  0.2  of  a  cubic  cen- 
timeter of  a  mixture  composed  of  2  cubic  centimeters  of  serum 
from  a  guinea-pig  immunized  against  rabbit  blood  and  previously 
heated  to  55  degrees,  and  20  drops  of  defibrinated  rabbit  blood.* 
In  other  words,  each  tube  receives  2  drops  of  well-sensitized  blood. 

The  result  of  the  experiment  is  as  follows: 

Hemolysis  takes  place  rapidly  and  nearly  simultaneously  in 
tubes  "b,"  "c,"  and  "d."  In  ten  minutes  no  intact  corpuscles 
remain.  In  tube  "a,"  containing  alexin,  bacilli  and  antiplague 
serum,  no  hemolysis  occurs.  The  corpuscles  also  remain  intact  in 
tubes  "e'7  and  "f,"  to  which  no  alexin  was  added.  We  find  then, 
first,  that  the  plague  bacillus  mixed  with  normal  horse  serum  does 
not  absorb  alexin;  and  second,  that  the  bacillus  with  antiplague 
serum  from  the  horse  does  absorb  alexin  with  avidity;  and  third, 
that  antiplague  serum  alone  has  no  effect  on  the  alexin. 

Consequently  we  must  conclude  that  the  serum  of  a  horse  vac- 

*  As  already  mentioned  in  previous  articles,  blood  that  has  been  washed  in  salt 
solution  is  generally  used  in  experiments  of  this  nature.  1  to  2  c.c.  of  defibrinated 
blood  is  placed  in  a  centrifuge  tube  and  the  level  marked  on  the  glass.  After 
centrifugalization  the  supernatant  fluid  is  drawn  up  with  a  bulb  pipette,  leaving 
the  deposited  corpuscles.  Enough  salt  solution  is  added  to  restore  the  blood  to  its 
original  level.  This  constitutes  defibrinated  blood  with  the  serum  replaced  by 
salt  solution,  in  other  words,  containing  no  alexin. 


THE  EXISTENCE  OF  SENSITIZING  SUBSTANCES.  221 

cinated  against  B.  pestis  contains  a  sensitizer  that  endows  this  bac- 
terium with  the  power  of  absorbing  alexin.  This  sensitizer  then  acts 
as  do  the  corresponding  substances  present  in  cholera  serum  and 
in  hemolytic  sera. 

It  may  be  added  that  plague  bacilli  added  to  a  mixture  of  alexin 
and  antiplague  serum  show  no  morphological  alteration  after  3  hours 
at  37  degrees.  Consequently  in  this  instance  the  presence  of  a 
sensitizer  can  be  demonstrated  only  by  the  fixation  of  the  alexin. 

If  in  such  an  experiment  much  smaller  doses  of  bacilli  and  anti- 
plague  serum  with  the  same  dose  of  alexin  are  employed,  a  complete 
fixation  does  not  take  place.  The  subsequently  introduced  cor- 
puscles are  finally  hemolyzed,  but  only  after  a  more  or  less  consider- 
able delay. 

The  serum  of  guinea-pigs  vaccinated  with  the  first  anthrax  vaccine. 
—  Guinea-pigs  were  given  four  successive  intraperitoneal  injections 
of  the  first  anthrax  vaccine.  Five-  to  six-day  pepton-bouillon  cul- 
tures were  used  for  the  first  two  injections.  Three-  or  four-day 
agar  cultures  suspended  in  salt  solution  were  used  for  the  last  two 
injections. 

An  experiment  similar  to  the  one  with  antiplague  serum  was  then 
performed.  As  a  control,  in  the  place  of  the  serum  of  the  vaccinated 
guinea-pigs,  normal  guinea-pig  serum  was  used.  As  a  bacterial 
emulsion  a  24-hour  agar  culture  of  the  first  vaccine  suspended  in 
salt  solution  was  used.  Fresh  normal  guinea-pig  serum  furnished 
the  alexin. 

The  results  were  identical  with  those  obtained  with  antiplague 
serum.  The  first  vaccine  plus  normal  serum  absorbs  little  or  no 
alexin.  There  is  complete  fixation  with  the  specific  serum.  In 
this  instance,  also,  the  fixation  of  alexin  causes  no  visible  lesion  of 
the  bacterium. 

Serum  of  a  horse  immunized  against  swine  plague. — Dr.  Frasey  of 
the  Pasteur  Institute  was  so  kind  as  to  furnish  us  with  strongly 
preventive  serum  for  the  swine-plague  bacillus.  In  this  serum  we 
were  also  able  by  the  same  method  to  demonstrate  a  sensitizer  which 
allows  the  swine-plague  bacillus  to  fix  alexin. 

Serum  of  guinea-pigs  vaccinated  against  B.  typhosus.  —  These 
guinea-pigs  were  given  three  injections  of  B.  typhosus  suspended 
from  agar  cultures  in  salt  solution.  An  experiment  modeled  after 


222  STUDIES  IN  IMMUNITY. 

the  others  showed  that  the  active  serum  of  these  guinea-pigs  causes 
an  energetic  fixation  of  alexin  by  B.  typhosus. 

It  seemed  worth  while  in  this  instance  to  determine  whether  this 
sensitizer,  that  causes  a  fixation  of  alexin,  is  strictly  specific.  For 
this  purpose  the  experimental  tubes  were  doubled  in  the  following 
manner:  One  series  of  tubes  contained  mixtures  of  normal  and 
specific  sera  with  or  without  the  emulsion  of  specific  bacilli,  as  has 
already  been  described.  In  a  second  series  of  tubes  the  amounts  of 
serum  were  identical  with  the  first  series,  but  an  emulsion  of  B. 
coli  instead  of  B.  typhosus  was  used.  Twenty-four-hour  agar 
cultures  of  each  organism  suspended  each  in  4  c.c.  of  salt  solution 
were  used.  The  agar  surface  covered  was  approximately  the  same 
with  each  organism;  and  yet  the  colon  suspension,  judging  from  its 
growth,  contained  more  organisms.  We  mention  this  point  so  that 
a  failure  of  B.  coli  to  fix  the  alexin  in  presence  of  antityphoid  serum 
may  not  be  attributed  to  an  insufficient  dose  of  bacteria. 

The  result  of  such  an  experiment  is  very  clear.  The  antityphoid 
serum  shows  very  marked  and  yet  not  absolute  specificity.  In 
fact  the  colon  bacillus  when  mixed  with  antityphoid  serum  acquires 
the  property  of  absorbing  alexin  to  a  certain  degree.  Whereas 
relatively  small  doses  of  the  typhoid  bacillus  and  antityphoid  serum 
absorb  alexin  completely,  much  larger  doses  of  B.  coli  with  the 
same  serum  are  required  to  produce  even  a  partial  fixation.  For 
example,  if  sensitized  blood  corpuscles  are  added  to  a  mixture  of 
0.2c.c.  of  guinea-pig  alexin,  0.2c.c.  typhoid  emulsion  and  O.Gc.c.  anti- 
typhoid serum  (56  degrees)  that  has  been  standing  for  a  few  hours, 
they  remain  indefinitely  intact;  in  a  mixture  containing  the  same 
amount  of  alexin  and  twice  the  dose  of  antityphoid  serum  and 
colon  emulsion,  the  same  dose  of  corpuscles  is  hemolyzed  after  an 
hour's  delay.  There  is  a  very  distinct  delay  in  this  tube,  as  the  con- 
trols containing  bacteria  are  completely  hemolyzed  in  15  minutes, 
and  those  without  bacteria  in  2  minutes. 

It  is  evident,  then,  that  the  colon  bacillus  reacts  distinctly,  though 
much  less  powerfully,  than  the  typhoid  bacillus  to  the  sensitizing 
effect  of  antityphoid  serum.* 

Serum  from  convalescent  typhoid  patients.  —  Dr.  Widal  has  been 

*  It  may  be  noted  that  this  serum  agglutinates  B.  typhosus  well,  but  has  no 
more  effect  on  B.  coli  than  normal  serum. 


THE  EXISTENCE  OF  SENSITIZING  SUBSTANCES.  223 

so  kind  as  to  allow  us  to  take  3  to  4  c.  c.  of  blood  from  two  of  the 
convalescent  cases  of  typhoid  in  his  hospital  service.  Both  these 
women  had  shown  the  classical  symptoms  of  typhoid  fever.  At 
the  time  the  blood  was  taken  the  temperature  had  been  normal  for 
from  20  to  30  days. 

The  sera  obtained  from  the  two  patients  were  heated,  together 
with  control  sera  from  each  of  us,  who  have  never  had  typhoid,  to 
56  degrees  for  a  half  hour.  A  small  amount  of  one  of  the  control 
sera  was  kept  unheated  for  alexin. 

The  result  of  the  experiment  was  very  convincing.  In  tubes 
containing  alexin  0.2  c.c.  (unheated  human  serum),  emulsion  of  B. 
typhosus  (0.5  c.c.)*  neither  of  our  heated  sera  caused  any  fixation 
of  the  alexin.  Sensitized  rabbit  corpuscles  added  a  few  hours  later 
were  hemolyzed  as  rapidly  as  in  control  tubes  containing  the  sera 
without  bacterial  emulsion.  In  the  tubes  containing  human  alexin, 
typhoid  bacillus,  and  either  of  the  heated  sera  from  typhoid  con- 
valescents, subsequently  added  sensitized  corpuscles  remained  in- 
tact for  days.  In  similar  mixtures  of  sera  without  bacilli,  hemolysis 
occurred  with  customary  rapidity. 

Consequently,  the  power  of  causing  the  typhoid  bacillus!  to 
absorb  human  alexin  is  very  marked  in  the  serum  of  patients  con- 
valescent from  typhoid. f 

It  would  be  of  interest  to  determine  just  how  highly  specific  such 
a  serum  is,  particularly  by  comparing  the  reaction  as  between  B. 
typhosus  and  B.  coli.  The  time  in  the  course  of  the  disease  at  which 
this  power  appears  in  the  serum  is  also  of  interest,  but  would  neces- 
sitate an  examination  of  a  large  number  of  cases.  Hitherto  we 
have  had  neither  the  time  nor  the  material  to  consider  these 
problems. 

Serum  of  guinea-pigs  vaccinated  against  B.  proteus  vulgaris. — The 
method  described  for  demonstrating  the  existence  of  a  sensitizer  by 

*  An  emulsion  prepared  by  suspending  a  24-hour  agar  culture  of  B.  typhosus 
in  5  c.c.  salt  solution  of  0.7  per  cent. 

t  The  culture  of  B.  typhosus  used  was  one  that  had  been  carefully  controlled 
by  Dr.  Binot  of  the  Pasteur  Institute,  who  was  so  kind  as  to  give  it  to  us. 

J  These  particular  sera  were  only  faintly  agglutinative  for  B.  typhosus.  In 
this  connection  the  observations  of  Pfeiffer  and  Kolle  may  be  recalled;  they  showed 
that  the  agglutinating  power  in  such  sera  does  not  run  parallel  to  the  bactericidal 
power. 


224  STUDIES  IN  IMMUNITY. 

fixation  of  alexin  is  superfluous  in  the  case  of  antiproteus  serum. 
The  proteus  bacillus  we  have  used  gives  a  granular  degeneration 
similar  to  the  one  shown  by  the  cholera  vibrio  in  cholera  serum 
when  placed  in  contact  with  fresh  normal  serum. 

We  know  that  the  vibrio,  too,  is  slightly  affected  by  normal  guinea- 
pig  serum.  Proteus  vulgaris  gives  a  complete  granular  transfor- 
mation with  a  sizable  dose  of  normal  serum,  but  a  much  smaller 
amount  suffices  if  a  little  fresh  or  heated  antiproteus  serum  is 
added.  This,  of  course,  demonstrates  the  presence  of  a  sensitizer 
in  the  immune  serum. 

But  we  have  also  done  the  alexin  fixation  experiment  with  Proteus 
vulgaris.  The  expected  result  was  obtained:  the  organism  in  the 
presence  of  normal  guinea-pig  serum  absorbs  little  or  no  alexin, 
but,  when  affected  by  the  specific  serum,  takes  it  up.  In  the  latter 
instance,  of  course,  sensitized  corpuscles  are  not  destroyed. 

In  the  experiments  up  to  this  point  we  have  uniformly  employed 
sensitized  rabbit  corpuscles  (i.e.,  those  treated  with  specific  serum 
from  the  guinea-pig  heated  to  55  degrees)  to  prove  the  presence  or 
absence  of  free  alexin  in  the  fluid.  In  view  of  the  conception  of 
the  unity  of  the  hemolytic  and  bacteriolytic  alexin  as  proved  a  year 
ago  we  might  have  used  as  an  indicator  either  other  corpuscles  or 
sensitized  bacteria,  such  as  cholera  vibrios,  to  show  the  presence  of 
free  alexin  by  a  morphological  change.  Such  an  experiment  we 
have  performed  with  B.  proteus  vulgaris  as  an  indicator: 

A  24-hour  agar  culture  of  Proteus  vulgaris  was  suspended  in 
6  c.c.  of  salt  solution.  An  agar  culture  of  the  cholera  vibrio  was 
treated  in  the  same  manner.  Fresh  normal  guinea-pig  serum,  as 
alexin,  and  heated  proteus  serum  and  cholera  serum  were  also  at 
hand.  The  following  mixtures  were  prepared: 

(a)  Alexic  serum,  0.2  c.c.;   proteus  emulsion,  0.3  c.c.;  proteus 
serum,  56  degrees,  0.6  c.c. 

(b)  Alexic  serum,  0.2  c.c.;  proteus  emulsion,  0.6  c.c.;  normal 
guinea-pig  serum,  56  degrees,  0.6  c.c. 

(c)  Same  as  "a,"  without  bacteria. 

(d)  Same  as  "b,"  without  bacteria. 

Five  hours  later  0.2  of  a  cubic  centimeter  of  the  following  mixtuiv 
was  added  to  each  tube:  Emulsion  of  cholera  vibrios  0.5  cubic 
centimeter,  cholera  serum,  1  cubic  centimeter  56  degrees  (from  a 


THE  EXISTENCE  OF  SENSITIZING  SUBSTANCES.  225 

vaccinated  guinea-pig).  After  1J  hours  in  the  thermostat  stained 
preparations  were  made  from  each  tube. 

In  tube  "a"  we  found  that  the  cholera  vibrios  kept  their  normal 
appearance,  but  all  the  Proteus  rods  showed  granular  degeneration. 
In  tube  "b,"  on  the  contrary,  numerous  Proteus  rods  were  found 
but  the  vibrios  had  all  lost  their  normal  appearance  and  become 
completely  metamorphosed.  In  tubes  "c"  and  "d,"  containing 
no  Proteus,  as  might  be  expected,  the  vibrios  were  completely 
transformed.*  It  is  obvious,  then,  that  in  tube  "a"  the  sensitized 
proteus  bacilli  have  absorbed  the  alexin  and  so  protected  the  sub- 
sequently added  vibrios. 

The  mixtures  were  left  at  room  temperature  overnight  and  the 
next  day  placed  in  the  incubator  for  6  hours.  When  stained  prepa- 
rations were  then  made  the  results  were  striking. 

In  tube  "a,"  in  which  the  proteus  bacilli  were  destroyed,  the 
cholera  vibrio  has  undergone  marked  multiplication.  In  tube 
"b,"  containing  non-sensitized  Proteus,  the  reverse  is  true;  the  Pro- 
teus has  grown  out,  but  no  vibrios  are  found.  Therefore  in  these 
two  tubes  containing  the  same  amount  of  alexin  the  bactericidal 
power  has  been  directed,  in  one,  against  one  organism,  in  the  other, 
against  another.  Although  the  vibrios  in  each  mixture  were  equally 
sensitized,  they  have  grown  in  tube  "a,"  because  the  harmful  in- 
fluence of  the  alexin  was  diverted  by  the  properly  sensitized  Pro- 
teus vulgaris;  this  bacillus  has  acted,  in  a  way,  as  a  shield  for  the 
vibrio.f 

The  data  considered  in  the  present  article,  when  added  to  those 
we  already  have,  give  the  conception,  that  under  immunization  an 
animal  forms  an  appropriate  sensitizer  capable  of  causing  the  bac- 
terium it  affects  to  absorb  alexin,  the  appearance  of  a  general 
law. 

We  shall  not  here  consider  how  far  the  presence  of  a  sensitizer  in 

*  In  controls  of  both  the  vibrio  and  Proteus  made  each  with  its  heated  specific 
serum  alone  there  is  agglutination,  but  no  metamorphosis. 

f  This  is  a  correct  demonstration  of  the  principle  established  by  one  of  us  in 
1895:  "That  the  bactericidal  substance  is  the  same  in  normal  as  in  vaccinated 
animals.  In  the  case  of  animals  vaccinated  against  certain  diseases  the  energy  of 
the  bactericidal  substance  acts  particularly  against  a  given  microbe  owing  to  the 
specific  preventive  substance  (sensitizer),  which  varies  according  to  the  bacterium 
used  for  immunization.  It  is  by  means  of  this  peculiar  preventive  substance  that 
the  animal  body  directs  its  destructive  power  against  a  particular  infection." 


226  STUDIES  IN   IMMUNITY. 

any  one  of  these  sera  endows  it  with  curative  or  preventive  proper- 
ties for  normal  animals.  We  may  say  simply  that  the  function  of 
sensitizers  in  protecting  animals  must  vary  with  the  bacteria  under 
consideration.  Certain  of  these  bacteria  are  easily  destroyed  in 
fixing  alexin,  others  under  the  same  conditions  resist  better  (leaving 
aside  any  purely  physiological  alteration  that  may  occur);  and 
others  doubtless  absorb  alexin  with  impunity.*  Several  therapeu- 
tic sera  owe  their  activity  in  great  part  to  the  presence  of  anti- 
toxins. The  seat  of  infection  must  enter  into  account,  as  alexin  is 
not  uniformly  distributed  throughout  the  body.  It  is  certain  that 
the  part  played  by  each  substance  of  active  serum  in  a  cure  by 
serum  therapy  must  vary  with  the  infection  under  consideration. 

There  is  one  fact  frequently  mentioned  in  this  article  that  we 
may  consider  for  a  moment.  The  bacteria  studied  had  little  or 
no  alexin-fixing  property  unless  sensitized,  and  even  when  sensitized 
the  bacteria  had  to  be  relatively  numerous  to  absorb  alexin.  Con- 
sequently, in  animals  invaded  by  a  pathogenic  micro-organism,  we 
should  scarcely  imagine  death  to  be  due  to  an  insufficiency  of  alexin 
in  the  body.  An  assertion  that  this  is  the  cause  of  death  is,  more- 
over, to  lose  sight  of  the  fundamental  and  well-proved  idea  that 
yielding  to  an  infection  is  primarily  due  to  an  inability  of  the  phag- 
ocytes to  take  up  the  infective  agent.  And  then  —  even  supposing 
protection  by  alexin  to  be  the  essential  factor  in  immunity  —  death 
would  not  be  due  to  a  lack  of  alexin,  but  rather  to  a  lack  of  utilizing 
or  absorbing  it. 

We  should  scarcely  expect  in  treating  human  bacterial  infections 
that  —  as  Wassermann  has  expressed  the  hope  —  the  administra- 
tion of  certain  normal  sera  or  alexins  in  addition  to  the  specific  serum 
could  be  recommended.  It  is  unreasonable,  since  the  alexins  from 
foreign  species  affect  not  only  bacteria,  but  body  cells.  And,  more- 
over, the  animal  would  soon  protect  itself  from  such  injections  by 
forming  anti-alexins. 

*  We  intend  to  determine  whether  the  serum  of  tuberculous  individuals  con- 
tains a  sensitizer  capable  of  causing  Koch's  bacillus  to  absorb  alexin.  If  such  a 
sensitizer  is  present,  it  would  be  a  good  example  of  one  that  serves  no  distinctly 
useful  purpose. 


THE  EXISTENCE  OF  SENSITIZING  SUBSTANCES.  227 

CONCLUSION: 

1.  Specific  sensitizers  are  formed  in  vaccinated  animals  as  a 
general  rule.    The  sensitizers  active  against  various  bacteria  have 
the  common  property  of  causing  the  organisms  they  affect  to  ab- 
sorb alexin. 

2.  The  extent  of  the  damage  that  alexin  absorption  causes  in 
bacteria  varies  with  the  organism  concerned. 


XI.    ON  THE  MODE  OF  ACTION  OF  CYTOLYTIC  SERA; 

AND  ON  THE  UNITY  OF  THE  ALEXIN  IN 

A  GIVEN  SERUM.* 

BY  DR.   JULES   BORDET. 

(From  Professor  Metchnikoff's  Laboratory.) 

The  conception  that  bacteriolysis  by  cholera  serum  and  that 
hemolysis  by  hemolytic  sera  are  due  to  the  combined  action  of 
two  distinct  substances,  as  proved  by  us  in  1895  and  1898,  is  now 
generally  accepted.  These  two  substances  are,  first,  the  alexin,  the 
cellulicidal  and  bactericidal  substance,  properly  speaking,  occurring 
in  the  serum  both  of  normal  and  of  immunized  animals;  and,  second, 
the  specific  sensitizing  substance  that  endows  an  immune  serum 
with  the  particular  property  of  favoring  alexic  activity.  In  collab- 
oration with  Dr.  Gengou  we  have  described  in  the  preceding  article 
a  method  of  demonstrating  the  existence  of  sensitizing  substances. 
This  method  depends  essentially  on  the  fact  that  a  specific  bac- 
teriolytic  or  hemolytic  serum  heated  to  55  degrees,  and  so  deprived 
of  its  own  destructive  power,  will  confer  a  bactericidal  or  hemolytic 
power  on  normal  alexic  serum  to  which  it  is  added.  We  further 
demonstrated  by  a  new  method  that  these  sensitizing  substances 
are  present,  certainly,  in  many,  and  probably  in  all,  antimicrobial 
sera  resulting  from  artificial  immunization. 

But  the  elementary  fact  that  the  destructive  action  of  a  serum 
is  due  to  the  collaboration  of  the  alexin  and  the  sensitizer  is  not 
sufficient.  An  attempt  should  be  made  to  discover  the  inner 
mechanism  of  the  phenomenon  and  to  determine  the  exact  nature 
of  the  reaction  between  the  active  substances  and  the  affected  cells. 
It  is  only  since  1899  that  this  new  problem  has  been  attentively 
considered;  the  data  obtained  are  not  as  yet  very  numerous  and 
yet  three  important  facts  have  been  firmly  established.  These 
facts  we  shall  enumerate  and  shall  then  consider  the  interpreta- 
tions to  which  they  have  given  rise. 

*  Sur  le  mode  d'action  des  scrums  cytolytique  et  sur  I'unitl  de  Palexine  dans 
un  meme  se>um.  Annales  de  I'lnstitut  Pasteur,  XV,  1901,  303. 

228 


MODE   OF  ACTION   OF  CYTOLYTIC  SERA.  229 

I.     DOES  THE  ALEXIN  UNITE  WITH  THE  SENSITIZING  SUBSTANCE? 

The  first  of  the  three  facts  mentioned  concerns  normal  sera  par- 
ticularly. In  many  instances  when  a  certain  amount  of  normal 
serum  from  one  species  is  added  to  the  red  blood  corpuscles  of 
another  species  only  slight  hemolysis  occurs.  It  is  particularly 
true  that  a  large  number  of  corpuscles  remain  intact  if  the  con- 
tact is  brief.  If  we  then  separate  these  corpuscles  by  centrifugaliza- 
tion,  and  wash  them  so  as  to  remove  the  normal  serum,  we  find  on 
adding  a  sensitizer  (that  is,  a  heated  serum  active  against  the  cor- 
puscles in  question)  that  no  hemolysis  occurs.  This  experiment 
was  first  performed  by  Ehrlich  and  Morgenroth.*  It  proves  that 
non-sensitized  corpuscles  mixed  with  normal  serum  do  not  absorb 
the  alexin,  for  we  know  that  if  the  alexin  is  present  the  addition 
of  an  appropriate  sensitizer  will  hemolyze  the  corpuscles.  This 
fact,  that  unsensitized  corpuscles  do  not  fix  the  alexin  of  normal 
serum,  is  applicable  only  to  those  corpuscles  that  have  remained 
intact,  but  bears  no  relation  to  the  few  corpuscles  that  have  been 
destroyed.  There  are  certain  normal  sera  that  have  great  hemo- 
lytic  activity  for  certain  species  of  corpuscles.  For  example^ 
hen  serum  destroys  rabbit  corpuscles  energetically.!  When  these 
corpuscles  are  added  to  hen  serum  they  absorb  alexin  very  dis- 
tinctly, as  is  shown  by  the  fact  that  fresh  rabbit  corpuscles  added 
to  such  a  hemolyzed  mixture  remain  intact.  J 

We  may  conclude  from  these  two  experiments  that,  when  mixed 
with  normal  serum,  corpuscles  that  remain  intact  do  not  affect 
the  alexin,  whereas  corpuscles  that  are  destroyed  absorb  a  certain 
amount. 

The  two  other  facts  about  the  specific  hemolytic  sera,  obtained 
by  injecting  animals  with  defibrinated  blood,  are  the  following: 

When  a  hemolytic  serum,  previously  heated  to  55  degrees,  is 
mixed  with  the  corpuscles  affected  by  that  serum,  these  corpuscles 

*  See  Collected  Studies  on  Immunity.  Ehrlich-Bolduan,  John  Wiley  and  Sons, 
p.  1. 

t   See  article,  p.  134. 

J  They  remain  intact  even  if  heated  hen  serum  is  also  added  to  the  mixture, 
which  proves  that  it  is  indeed  the  alexin  that  has  been  absorbed.  The  objection 
indeed  might  be  raised  that  the  first  corpuscles  destroyed  have  not  absorbed  much 
alexin,  but  have  fixed  the  normal  sensitizer  of  hen  serum  necessary  for  a  subsequent 
hemolysis;  the  addition  of  heated  hen  serum  answers  this  objection. 


230  STUDIES  IN  IMMUNITY. 

absorb  the  sensitizer  energetically  and  deprive  the  fluid  of  it.  This 
fact  was  first  irrefutably  proved  by  Ehrlich  and  Morgenroth.* 

The  third  fact  established  by  us  a  year  ago  f  is  that,  in  a 
mixture  of  fresh  normal  serum  (alexin)  and  sensitized  corpuscles 
or  bacteria  (that  is,  treated  with  an  appropriate  heated  hemo- 
lytic  or  bacteriolytic  serum)  the  alexin  that  destroys  the  sensitized 
cells  is  absorbed  by  them  and  disappears  from  the  fluid.  This  fixa- 
tion may  be  so  complete  that  the  fluid  completely  loses  its  power 
to  destroy  subsequently  added  sensitized  cells. 

These  are  the  principal  facts  derived  from  experiments,  apart 
from  any  theory.  We  may  now  attempt  to  explain  the  reactions 
between  sensitive  cells  and  active  substances  in  the  light  of  these 
facts. 

According  to  Ehrlich  and  Morgenroth  the  specific  antibody 
(sensitizer)  acts  as  a  real  intermediary  body  (Zwischenkorper, 
Ambocep tor),  a  joining  link  united  on  the  one  hand  to  the  corpuscles 
and  on  the  other  to  the  alexin.  In  other  words,  the  absorption  of 
the  alexin  by  the  sensitized  corpuscles  is  not  due  to  any  distinct 
affinity  of  the  corpuscles  for  the  alexin.  The  absorption  of  the 
alexin  is  indirect  only:  the  corpuscle  is  joined  to  the  intermediary 
substance  which  unites  chemically  by  its  other  pole  with  the  alexin. 

Our  conception  of  the  phenomenon  is  quite  different.  We  re- 
gard the  sensitizer  as  uniting  with  the  corpuscle  and  so  modifying 
it  as  to  allow  a  direct  absorption  of  the  alexin.  The  action  of  the 
sensitizer  on  cells  would  be  similar  to  that  of  fixing  or  mordanting 
agents  that  give  certain  substances  or,  in  histological  technic,  certain 
cells  the  power  of  absorbing  dyes  that  were  previously  not  taken. 
We  know  that  slight  modifications  suffice  to  make  cells  take  stains 
that  normally  do  not  affect  them.  To  be  sure,  in  speaking  of  mor- 
danting we  do  not  mean  that  all  the  phenomena  of  dyeing  must 
agree  with  the  phenomena  under  consideration;  we  have  simply 
offered  a  comparison  in  order  to  simplify  our  explanation.  The 
hypothesis  we  wish  to  emphasize  is  that  in  presence  of  hemolytic 
serum  the  corpuscle  itself  becomes  capable  of  absorbing  alexin 
directly,  owing  to  its  modification  by  the  sensitizer.  In  other  words, 
we  see  no  reason  for  considering  that  the  sensitizer  itself  combines 

*  Loc.  cit.,  p.  6. 
t  See  p.  186. 


MODE  OF  ACTION   OF  CYTOLYTIC  SERA.  231 

with  the  alexin,  as  Ehrlich  and  Morgenroth  believe,  or  that  this 
union  is  necessary  in  order  that  the  alexin  may  reach  the  corpuscle. 

It  must  be  admitted  that  both  of  these  interpretations  are  purely 
hypothetical  and  have  been  sanctioned  by  no  well-proved  fact.  We 
should  then  find  out  how  far  either  of  these  hypotheses  conforms 
with  reality  by  determining  how  far  deductions  from  them  agree 
with  experimental  fact. 

Let  us  consider  a  fresh  hemolytic  serum  containing  both  alexin 
and  sensitizer.  According  to  Ehrlich  and  Morgenroth,  this  sen- 
sitizer  is  combined  with  alexin — if  not  with  all  of  it,  owing  to  its 
possible  excess,  at  least  with  a  more  or  less  considerable  amount 
of  it.  When  corpuscles  are  added  the  sensitizer  unites  with  them, 
dragging  after  it  the  alexin  fixed  by  its  other  pole.  The  amount 
of  alexin  that  can  affect  the  corpuscles  must  be  only  that  portion 
previously  fixed  by  the  sensitizer.  If  there  is  more  alexin  in  the 
serum  than  is  necessary  to  saturate  the  sensitizer,  the  excess  could 
have  no  effect  on  the  corpuscles  and  would  consequently  not  be 
utilized.  We  may  therefore  conclude  that  the  added  corpuscles 
are  unable  to  modify  in  the  slightest  degree  the  established  relations 
between  alexin  and  sensitizer;  they  simply  fix  the  sensitizer  and  are 
destroyed  by  the  alexin  already  united  with  this  intermediary 
body. 

What  will  happen  if  we  add  to  such  an  active  hemolytic  serum, 
specific,  let  us  say,  for  rabbit  corpuscles,  another  sensitizer,  say  a 
heated  serum  affecting  hen  corpuscles?  The  result  is,  as  might  be  an- 
ticipated, that  the  mixture  obtained  destroys  hen  or  rabbit  corpuscles 
indifferently.  In  accordance  with  the  theory  of  Ehrlich  and  Mor- 
genroth one  must  consider  the  two  sensitizers  as  struggling  to  share 
the  alexin;  part  of  it  unites  with  sensitizer  A  and  the  rest  with 
sensitizer  B.  If  we  then  add  the  corpuscles  affected  by  sensitizer 
A,  obviously  they  will  absorb  it  and  be  injured  by  the  alexin  already 
united  to  this  intermediary  body  A.  But  there  is  no  logical  reason 
to  suppose  that  these  corpuscles  will  be  attacked  by  the  rest  of  the 
alexin  united  to  the  other  sensitizer  that  has  no  combining  influ- 
ence with  them.  Consequently,  on  adding  subsequently  the  other 
species  of  corpuscles,  we  should  expect  them  to  be  destroyed  by  the 
remainder  of  the  alexin  attached  to  sensitizer  B  which  is  specific 
for  these  corpuscles. 


232  STUDIES  IN  IMMUNITY. 

Such  an  experiment  planned  to  corroborate  these  theoretical 
deductions  from  the  Ehrlich  and  Morgenroth's  hypothesis  fails 
completely  to  do  so.  Two  exactly  similar  mixtures  A  and  B  are 
prepared,  containing  each  0.2  of  a  cubic  centimeter  of  fresh  hemo- 
lytic  serum  from  a  guinea-pig  that  had  been  given  four  injections 
(from  4  to  5  c.c.)  of  rabbit  blood,  and  1  c.c.  of  the  heated  (55 
degrees)  serum  of  a  rabbit  treated  with  hen  blood.  To  mixture 
A  is  added  0.6  of  a  cubic  centimeter  of  defibrinated  washed  hen 
blood;  the  corpuscles  are  soon  destroyed.  Nothing  is  added  to 
mixture  B  for  the  moment. 

A  few  hours  later  2  drops  of  defibrinated  rabbit  blood  are  added 
to  each  mixture.  In  mixture  B  destruction  of  the  corpuscles  is 
complete  in  about  45  minutes.  There  is  then  enough  alexin  present 
to  destroy  these  corpuscles;  we  have,  moreover,  admitted  that  the 
portion  of  the  alexin  that  produces  this  effect  was  already  com- 
bined with  the  sensitizer  for  the  corpuscles  in  question.  The  other 
mixture,  however,  proves  that  this  is  not  so. 

Mixture  A  is  identical  with  B  except  it  contains  in  addition  hemo- 
lyzed  hen  corpuscles.  The  rabbit  corpuscles  remain  intact  in  this 
mixture;  even  on  the  following  day  they  may  be  discerned  among 
the  nuclei  of  the  hen  corpuscles.*  The  hen  corpuscles  then  have 
used  up  all  the  alexin,  and  if  we  were  to  follow  the  hypothesis  of 
Ehrlich  and  Morgenroth,  we  should  be  forced  to  conclude  that  the 
sensitizer  acting  on  hen  corpuscles  had  combined  with  all  the  alexin, 
preventing  the  other  sensitizer  (active  against  rabbit  corpuscles) 
from  sharing  it.  Such  a  conclusion  must  also  hold  for  mixture  B 
containing  the  same  doses  of  the  same  sera.  And  yet  in  this  mix- 
ture B  the  sensitizer  that  has  not  combined  with  the  alexin  has 
been  able  to  destroy  its  appropriate  corpuscles.  There  is  no  reason, 
then,  in  explaining  hemolysis,  to  assume  that  the  sensitizer  combines 
with  the  alexin.  On  the  contrary,  it  seems  clear  to  us  from  this 
experiment  that  the  corpuscles  modified  by  their  union  with  the 
sensitizer  absorb  the  alexin  directly  and  prevent  it  from  acting  on 
other  cells.  When  the  corpuscle  is  not  present  the  sensitizer  in 
no  way  fixes  the  alexin  or  prevents  it  from  acting  on  the  first  properly 

*  The  destruction  of  the  rabbit  corpuscles  takes  place  on  addition  of  alexin 
(normal  guinea-pig  serum) ;  the  corpuscles  are  then  rapidly  destroyed,  showing 
that  they  have  absorbed  their  appropriate  sensitizer. 


MODE  OF  ACTION  OF  CYTOLYTIC  SERA.  233 

sensitized  cell  with  which  it  comes  in  contact.  It  would  be  well, 
then,  to  give  up  the  terms  Zwischenkorper,  Amboceptor,  and  Com- 
plement, words  chosen  under  the  domination  of  theories  which, 
although  ingenious,  and  capable  of  having  advanced  science 
by  the  experiments  they  have  suggested,  are  not  justified  by 
experiment.* 

This  experiment  we  have  just  quoted  is  similar  to  those  we  have 
already  discussed  in  previous  articles.  We  wish  here  simply  to 
emphasize  more  fully  the  conclusions  that  may  be  drawn  from 
them;  it  also  seems  wise  to  repeat  the  experiment  with  certain 
variations  to  show  tnat  it  invariably  gives  the  same  results. 

For  example,  we  can  make  a  mixture  of  0.5  of  a  cubic  centi- 
meter of  fresh  unheated,  normal  guinea-pig  serum,  2.5  c.c.  of 
heated  serum  from  a  guinea-pig  immunized  against  rabbit  blood 
and  2.5  c.c.  of  heated  serum  from  a  guinea-pig  immunized  against 
hen  blood.  One-tenth  of  a  cubic  centimeter  of  this  mixture,  con- 
taining alexin  and  the  two  sensitizers,  destroys  0.1  of  a  cubic  centi- 
meter of  defibrinated  washed  hen  blood.  The  hen  blood,  however, 
remains  intact  in  twice  the  amount  of  mixture,  0.2  of  a  cubic 
centimeter,  if  there  has  previously  been  added  to  it  0.3  of  a  cubic 
centimeter  of  washed  rabbit  blood.  The  rabbit  corpuscles  are 
destroyed  and  absorb  all  the  alexin  present  without  leaving  any  for 
the  hen-corpuscle  sensitizer. 

In  a  similar  mixture  of  fresh  normal  guinea-pig  serum,  heated 
guinea-pig  serum  affecting  rabbit  blood,  and  heated  rabbit  serum 
active  against  hen  corpuscles,  the  reverse  experiment  may  be  per- 
formed. This  mixture  is  divided  in  equal  parts.  To  one  is  added 
corpuscles  A  and  a  few  hours  later  corpuscles  B ;  to  the  other,  first 
B,  then  A.  As  in  the  preceding  experiments,  the  introduction  of 

*  In  the  experiment  just  considered  there  is  a  third  mixture  C  containing,  as  the 
others,  0.2  of  a  cubic  centimeter  of  fresh  hemolytic  serum  specific  for  rabbit  cor- 
puscles. Instead  of  heated  rabbit  serum  active  against  hen  corpuscles  it  contains 
the  same  amount  (1  c.c.)  of  heated  normal  rabbit  serum.  When  0.6  of  a  cubic 
centimeter  of  hen  blood  is  added  there  is  no  hemolysis.  On  adding  2  drops  of 
rabbit  blood  hemolysis  is  as  rapid  as  in  B.  Since  B  and  C  act  alike  we  conclude 
that  neither  the  sensitizer  for  hen  corpuscles,  nor  hen  corpuscles  themselves,  are 
able  alone  to  absorb  alexin;  the  two  must  be  united  to  produce  this  result.  As 
we  have  already  shown  in  a  similar  experiment  (see  p.  186),  corpuscles  from 
species  other  than  the  one  affected  by  the  sensitizer  that  is  present  leave  the 
alexin  unchanged. 


234  STUDIES  IN  IMMUNITY. 

one  kind  of  corpuscle  prevents  destruction  of  the  second  kind. 
Controls  show  that  the  amount  of  either  species  of  corpuscles  added 
is  rapidly  destroyed  with  even  half  the  amount  of  each  mixture 
provided  that  the  other  kind  of  corpuscles  has  not  previously  been 
added. 

The  experiments  reported  in  collaboration  with  Dr.  Gengou  in 
the  previous  article  are  also  of  significance  in  this  connection.  They 
prove  that  the  addition  to  alexic  serum  of  a  sensitizer  active  against 
bacteria  does  not  deprive  the  serum  of  its  power  to  destroy  sub- 
sequently added  sensitized  corpuscles  or  sensitized  bacteria  of 
another  Variety.  But  if  the  sensitizer  added  in  the  first  place  is 
accompanied  by  the  bacteria  for  which  it  is  specific,  the  serum  is 
entirely  deprived  of  its  alexic  power.  Here  again  it  is  the  sensi- 
tizer plus  bacterium  and  not  the  sensitizer  alone  that  takes  up 
the  alexin. 

We  may  now  consider  an  objection  that  Ehrlich  and  Morgenroth 
have  raised  to  our  hypothesis.  These  investigators  imagine  that 
to  agree  with  our  conception  a  sensitized  corpuscle  should  be  equally 
affected  by  any  alexin  coming  from  any  animal  species.  They 
mention  the  fact  that  rabbit  corpuscles  must  be  much  more  power- 
fully sensitized  to  be  hemolyzed  by  rabbit  alexin  than  when  guinea- 
pig  alexin  is  employed. 

The  fact  is  exact;  we  are  all  the  more  ready  to  admit  it  as  we 
ourselves  mentioned  it  in  the  article  that  gave  rise  to  Ehrlich  and 
Morgenroth's  objection.*  We  are  at  a  loss,  however,  to  understand 
how  these  investigators  should  imagine  that  in  our  opinion  sen- 
sitized corpuscles  should  be  equally  susceptible  to  various  alexins. 
Our  idea  is  quite  the  opposite.  Since  the  alexins  in  the  different 
animal  species  are  not  identical,  it  is  quite  obvious  that  they  should 
not  all  have  an  equal  tendency  to  destroy  a  given  corpuscle.  And 
consequently  a  weak  sensitization  may  at  times  render  a  corpuscle 
susceptible  to  certain  alexins,  while  a  strong  sensitization  would  be 
necessary  to  cause  weaker  alexins  to  produce  as  great  an  effect. 
In  the  instance  under  consideration,  it  is  quite  conceivable  that 
rabbit  corpuscles  must  be  strongly  sensitized  to  yield  to  rabbit  alexin. 
which,  under  normal  conditions,  is  quite  harmless  for  its  proper 
corpuscles.  It  is  evident,  then,  that  a  given  dose  of  a  given  sensi- 

*  See  p.  187. 


MODE  OF  ACTION   OF  CYTOLYTIC  SERA.  235 

tizer  would  seem  to  affect  a  corpuscle  differently,  according  to  the 
alexin  used.  In  different  instances  the  sensi tizer  may  be  the  same, 
but  the  dose  would  vary  with  the  alexin  employed. 

From  this  easily  explicable  fact,  that  the  amount  of  sensitizer 
necessary  varies  with  the  alexin  employed,  Ehrlich  and  Morgenroth 
feel  justified  in  concluding  that  the  serum  contains  several  sen- 
sitizers, all  of  which  affect  rabbit  corpuscles,  but  which  differ  from 
one  another.  One  of  these  sensitizers  renders  the  corpuscle  suscep- 
tible to  guinea-pig  alexin,  and  another  to  rabbit  alexin.  This  com- 
plicates unnecessarily  the  already  sufficiently  complicated  question 
of  hemolytic  sera.* 

II.    ON  THE  UNITY  OF  THE  ALEXIN  IN  A  GIVEN  SERUM. 

We  know  that  the  alexins  from  different  animal  species  are  not 
identical.  But  does  a  given  alexic  serum,  say  from  the  guinea-pig, 
contain  a  single  alexin  or  several  of  them  that  differ  in  chemical 
constitution?  It  is  rather  difficult  to  reply  to  the  question  put  in 
this  way,  as  our  knowledge  of  the  chemical  nature  of  the  alexin 
is  practically  nil.  We  may  question,  however,  whether  the  alexin  is 
functionally  simple,  that  is  to  say,  whether  the  alexin  (or  alexins, 
if  there  are  several) 'can  attack  various  cells  such  as  corpuscles  and 
bacteria  indifferently,  particularly  when  these  cells  are  sensitized. 
In  this  form  the  question  is  of  lively  interest  in  studies  on  immunity 
and  may  be  more  easily  answered. 

We  know  that  Buchner  in  his  first  studies  on  the  alexins  of  normal 
sera,f  that  have  by  now  become  classical,  considered  the  substance 
that  destroys  bacteria  as  identical  with  the  one  that  produces 
hemolysis.  We  offered  facts  a  year  ago  that  seemed  to  corroborate 
this  opinion. 

We  have  just  recalled  these  facts  in  the  preceding  article.  When 
a  serum  is  deprived  of  its  alexin  by  the  addition  of  a  sensitized 
bacterium  or  corpuscle,  the  fluid  becomes  incapable  of  destroying 
another  sensitized  cell,  even  when  it  is  different  from  the  one  that 
has  taken  up  the  alexin  in  the  first  place.  It  must  then  be  always 

*  By  the  same  process  of  reasoning  Ehrlich  and  Morgenroth  think  that  in  a 
given  normal  serum  there  are  several  intermediary  bodies  (sensitizers),  all  active 
against  the  same  corpuscles,  but  each  requiring  a  different  alexin. 

t  See,  for  example,  Verhandlungen  der  Congresses  fur  innere  Medicin,  1892. 


23G  STUDIES  IN   IMMUNITY. 

the  same  alexin  that  unites  with  and  destroys  various  cells.  In 
the  preceding  article  with  Dr.  Gengou  we  found  that  various 
bacteria  absorb  the  alexin  which  is  essential  for  the  destruction 
of  red  blood  cells  or  of  other  bacteria. 

We  may  mention  two  other  similar  experiments.  The  red  spiril- 
lum, a  non-pathogenic  organism,  is  readily  destroyed  by  the  serum 
of  a  normal  guinea-pig,  even  without  the  addition  of  a  sensitizer. 
In  each  of  two  tubes  is  placed  0.2  of  a  cubic  centimeter  of  fresh 
hemolytic  serum  obtained  from  guinea-pigs  immunized  against  rab- 
bit blood.  To  tube  "a"  0.3  of  a  cubic  centimeter  of  guinea-pig 
blood  is  added;  to  tube  "b"  0.3  of  a  cubic  centimeter  of  washed 
rabbit  blood.  After  a  few  hours  2  drops  of  an  emulsion  of  the 
spirillum  are  added  to  each  tube  and  they  are  placed  in  the  incu- 
bator. The  spirilla  are  destroyed  in  tube  "  a, "  in  which  the  guinea- 
pig  corpuscles  have  remained  intact;  they  remain  normal  in  tube 
"b"  containing  hemolyzed  rabbit  corpuscles.  Sensitized  hen  cor- 
puscles subsequently  added  to  both  tubes  are  hemolyzed  in  "a, " 
but  remain  intact  in  "b."  Rabbit  corpuscles,  then,  by  absorbing 
the  alexin  protect  two  very  different  cells  —  the  spirillum  and  hen 
corpuscles. 

In  the  other  experiment  we  have  to  deal  with  a  rather  peculiar 
instance.  We  may  ask  whether  the  rabbit  alexin  that  destroys  sen- 
sitized rabbit  corpuscles  differs  from  the  alexin  in  the  same  serum 
which  attacks  other  species  of  corpuscles.  Experimentally  we  find 
that  it  does  not.  In  each  of  two  tubes  "a"  and  "b"  is  placed 
0.2  of  a  cubic  centimeter  of  washed  hen  blood;  0.2  of  a  cubic  cen- 
timeter of  fresh  normal  rabbit  serum  is  then  added  to  each.  To 
tube  "a"  is  then  added  0.6  of  a  cubic  centimeter  of  serum  from  a 
rabbit  treated  with  hen  blood  and  heated  to  56  degrees.  To  tube 
"b"  0.6  of  a  cubic  centimeter  of  normal  rabbit  serum  (56  degrees) 
is  added.  The  hen  corpuscles  are  hemolyzed  in  "a,"  but  remain 
intact  in  "b."  After  a  few  hours  0.2  of  a  cubic  centimeter  of  the 
following  mixture  is  added  to  each  tube:  Washed  rabbit  blood, 
20  drops;  serum  of  a  guinea-pig  treated  with  rabbit  blood  and 
heated  to  56  degrees,  2  c.c. 

These  sensitized  corpuscles  are  destroyed  in  "b"  and  remain 
intact  in  "a. " 

The  fact  that  a  given  alexin  may  attack  different  cells  seems  to 


MODE  OF  ACTION  OF  CYTOLYTIC  SERA.  237 

us  sufficiently  proved  by  all  these  experiments.*  We  must  now 
take  up  certain  objections  that  have  been  offered  to  this  opinion, 
some  of  which  seem  rather  serious. 

Ehrlich  and  Morgenroth  found  that  the  serum  of  a  goat  immu- 
nized against  sheep  blood,  when  heated  to  56  degrees  or  even  higher, 
loses  its  power  of  destroying  various  species  of  corpuscles,  such  as 
the  guinea-pig  and  the  rabbit,  which  it  previously  attacked.  This 
loss  affects  the  non-specific  hemolytic  power  present  in  normal 
goat  serum  and  acting  on  corpuscles  not  used  in  the  immunization 
of  this  animal.  But  this  goat  serum  still  shows  a  distinct  ability 
to  destroy  sheep  blood,  for  which  it  is  specific.  Ehrlich  and  Mor- 
genroth conclude  that  this  serum  contains  several  alexins,  some 
of  which  are  destroyed  at  56  degrees  and  others  of  which  resist  this 
temperature.  It  is  just  as  easy  to  imagine  that  we  have  to  deal 
with  a  single  alexin  modified  or  attentuated  by  heating.  The 
alteration  is  sufficient  to  inhibit  the  hemolysis  of  non-sensitized 
corpuscles,  which  is  never  very  strong,  but  does  not  prevent  the 
destruction  of  corpuscles  that  are  sensitized  and  so  rendered  more 
susceptible  by  the  same  serum. 

In  another  experiment  Ehrlich  and  Morgenroth  pass  the  serum 
of  a  normal  goat,  that  destroys  guinea-pig  and  rabbit  corpuscles, 
through  a  Pukal  filter.  After  filtration  the  serum  still  destroys 
guinea-pig  corpuscles,  but  does  not  affect  rabbit  corpuscles.  Accord- 
ing to  these  authors  one  of  the  alexins  is  retained  by  the  filter  and 
the  other  passes  through ;  this  would  imply  that  the  two  alexins  in 
question  must  have  well-marked  chemical  differences  to  act  so 
differently  in  respect  to  the  filter.  It  is  to  be  noted  that  here  again 
we  are  dealing  with  hemolysis  by  a  normal  serum  which  is  never 
intense  and  may  be  affected  by  slight  variations.  It  is  quite  evi- 
dent that  the  serum  after  filtration  does  not  have  as  much  alexin 
as  before,  but  there  is  no  reason  for  supposing  that  there  are  two 
distinct  alexins.  It  may  well  be  imagined  that  filtration,  by  remov- 
ing from  serum  some  of  its  elements,  has  so  modified  its  physical 
properties  as  to  render  it  less  favorable  for  preserving  corpus- 
cles; we  may  well  imagine,  then,  that  certain  species  of  corpuscles, 

*  It  is  also  in  harmony  with  the  fact  we  previously  established,  that  an  anti- 
alexic  serum,  neutralizing  the  alexin  of  the  serum  of  a  given  species,  protects  the 
various  cells  that  are  affected  by  this  alexin,  even  when  they  are  sensitized. 


238  STUDIES  IN  IMMUNITY. 

which  are  more  susceptible  than  others  to  the  physical  changes 
in  the  surrounding  fluid,  should  yield  more  readily  to  traces  of 
alexin.  However  this  may  be,  this  experiment  does  not  deal 
with  sensitization  by  specific  serum  and  therefore  does  not  affect 
our  thesis  that  the  same  alexin  can  destroy  various  sensitized  cells 
energetically.* 

Neisser,t  a  partisan  of  the  plurality  of  alexins  in  a  given  serum, 
emphasizes  experiments  both  by  BailJ  and  by  himself,  the  prin- 
ciple of  which  is  as  follows :  if  a  normal  serum  is  mixed  with  cell  A 
(bacterium  or  blood  cell),  and,  after  a  sufficient  contact,  centrifu- 
galized,  it  is  found  that  the  supernatant  serum  has  lost  its  property 
of  destroying  cell  A,  but  will  still  destroy  cells  B  or  C.  Neisser 
concludes  from  this  that  the  alexin  affecting  A  is  not  identical  with 
the  one  attacking  B  or  C. 

The  experiment  contains  a  serious  experimental  error  and  con- 
sequently any  conclusion  from  it  is  erroneous.  In  the  majority 
of  instances,  when  even  large  amounts  of  non-sensitized  cells  are 
mixed  with  normal  serum,  they  absorb  only  a  small  amount  of  alexin. 
Our  experiments  just  related  as  well  as  those  with  Gengou  prove 
this  fact  conclusively.  Under  these  conditions  the  fluid  still  retains 
its  property  of  destroying  subsequently  added  sensitized  cells.  It 
is  reasonable  to  expect,  therefore,  that  if  an  unsensitized  or  feebly 
sensitized  cell  is  added  to  normal  serum,  for  example  corpuscles 
of  species  A,  there  will  soon  be  a  state  of  equilibrium  established 
and  a  division  of  the  alexin  between  the  cell  and  the  fluid;  the 
fluid,  however,  will  retain  the  greater  part.  Hemolysis,  moreover, 
never  goes  beyond  a  certain  point.  It  is  quite  possible  that  de- 
stroyed corpuscles  liberate  a  substance  that  prevents  the  fluid  from 
acting  further  on  corpuscles  of  the  same  variety,  without  inhibiting 
its  effect  on  other  varieties  of  corpuscles.  In  other  words,  a  state 
of  equilibrium  for  corpuscle  A  may  be  far  from  being  so  for  another 
cell,  which,  when  subsequently  added,  is  destroyed  by  the  fluid. 

In  view  of  these  facts  we  should  not  feel  justified  in  saying  that 

*  The  experiment,  indeed,  does  not  inform  us  as  to  whether  the  filtered  serum 
would  have  a  varying  hemolytic  power  for  two  different  species  of  corpuscles, 
each  sensitized  with  specific  heated  serum. 

t  Ueber  die  Vielheit  der  in  normalen  Serum  vorkommenden  Antikorper. 
Deutsche  med.  Wochen.,  1900,  No.  49. 

t  Archiv.  fur  Hygiene,  1899,  Bd.  XXV. 


MODE   OF  ACTION  OF  CYTOLYTIC  SERA.  239 

even  after  a  prolonged  contact  (16  hours)  normal  guinea-pig 
serum  (3  c.c.)  mixed  with  defibrinated,  carefully  washed  rabbit 
blood  (6  c.c.)  has  lost  all  of  its  alexin  for  rabbit  corpuscles.  The 
supernatant  fluid  from  such  a  mixture  after  centrifugalization  is 
very  red*  and  has  no  effect  on  freshly  added  rabbit  corpuscles;  in 
other  words,  it  has  become  inactive  for  these  corpuscles;  but  it  will 
still  produce  a  distinct  hemolysis  of  hen  corpuscles. 

We  may  be  sure  that  this  fluid  still  contains  a  definite  amount 
of  alexin,  to  the  presence  of  which  is  due  the  destruction  of  hen 
corpuscles.  As  far  as  rabbit  corpuscles  are  concerned,  a  state  of 
equilibrium  in  the  division  of  the  alexin  has  been  established  (and 
in  producing  this  state  the  products  from  destroyed  rabbit  cor- 
puscles have  perhaps  contributed),  and  further  attack  on  the  same 
corpuscles  thereby  prevented. 

This  state  of  equilibrium  is  easily  broken:  it  suffices  to  increase 
by  means  of  a  sensitizer  the  avidity  of  the  rabbit  corpuscles  for 
alexin.  The  reddish  fluid  under  consideration  will  still  destroy 
fresh  rabbit  corpuscles  energetically  if  they  are  sensitized.  In  a 
mixture  of  0.2  of  a  cubic  centimeter  of  red  fluid,  the  same  amount 
of  washed  rabbit  blood,  and  0.6  of  a  cubic  centimeter  of  heated 
specific  guinea-pig  serum,  hemolysis  occurs  rapidly  and  completely. 
To  render  the  experiment  more  complete  another  mixture  is  pre- 
pared, containing,  in  the  place  of  the  sensitizing  serum,  0.6  of  a  cubic 
centimeter  of  heated  normal  guinea-pig  serum  (56  degrees).  In 
this  mixture  there  is,  of  course,  no  destruction  of  the  rabbit  corpus- 
cles. A  few  hours  later  0.3  of  a  cubic  centimeter  of  specific  anti- 
hen  serum  (56  degrees)  plus  0.1  of  a  cubic  centimeter  of  defibri- 
nated  washed  hen  blood  is  added  to  each  tube.  The  hen  corpuscles 
are  rapidly  destroyed  in  the  second  tube,  but  remain  quite  intact  in 
the  first. 

It  is  evident,  then,  that  the  normal  alexic  serum  has  by  no  means 
been  exhausted  by  the  long  contact  with  an  excess  of  rabbit  cor- 
puscles, but  has  retained  a  considerable  amount  of  the  alexin  fitted 
to  destroy  these  same  corpuscles.  If  the  rabbit  corpuscles  are 
sensitized,  the  absorption  of  alexin  takes  place  much  more  energeti- 
cally, and  new  corpuscles  of  another  species  subsequently  intro- 
duced remain  unchanged. 

*  There  are  many  intact  rabbit  corpuscles  present  in  the  sediment. 


240  STUDIES  IN   IMMUNITY. 

This  criticism  of  Neisser's  experiments  is  significant :  it  indicates 
that  much  prudence  should  be  exercised  in  drawing  conclusions 
from  similar  experiments  dealing  with  normal  sera;  the  multiplicity 
of  active  substances  in  such  sera  have  been  too  hastily  presumed. 
We  feel  that  it  is  the  more  permissible  for  us  to  venture  this  asser- 
tion, as  it  is  perhaps  applicable  also  to  a  certain  experiment  of  our 
own  on  the  multiplicity  of  agglutinins  in  normal  horse  serum.  At 
any  rate  the  subject  is  still  far  too  obscure  to  allow  of  any  exact 
opinion  on  substances  other  than  the  alexins. 

In  this  chapter  we  have  considered  only  the  question  of  the  unity 
or  plurality  of  the  alexin  (Ehrlich  and  Morgenroth's  complement) 
in  a  given  serum.  We  have  not  sought  to  invalidate  the  idea, 
drawn  from  certain  experiments  of  Ehrlich  and  Morgenroth*  in 
particular,  that  normal  sera  may  contain  in  addition  to  the  alexin 
(complement)  one  or  several  normal  sensitizers  which,  although 
weaker  than  those  in  specific  sera,  facilitate  the  activity  of  the 
alexin. 

*  These  experiments  indeed  agree  with  our  own  (p.  97),  that  indicate  the 
existence  of  a  substance  in  normal  horse  serum  that  sensitizes  the  cholera  vibrio 
to  a  certain  extent  to  the  alexin  of  another  normal  serum. 


XII.   ON  THE  SENSITIZERS  OF  SERA  ACTIVE  AGAINST 
ALBUMINOUS  SUBSTANCES  * 

BY  DR.   OCTAVE   GENGOU. 

The  experimental  study  of  anticholera  immunity  in  vivo  led 
Pfeifferf  to  discover  the  phenomenon  that  now  bears  his  name, 
which  consists  in  an  extracellular  granular  transformation  of 
Koch's  vibrios  in  the  peritoneal  cavity  of  guinea-pigs  vaccinated 
against  cholera.  The  same  phenomenon  also  occurs  in  the 
peritoneum  of  normal  guinea-pigs,  as  Pfeiffer  himself  showed,  on 
injecting  cholera  vibrios  mixed  with  serum  from  well-immunized 
animals. 

This  destructive  transformation  of  vibrios  indicates  an  energetic 
bactericidal  action.  Pfeiffer  attributed  to  the  fixed  endothelial 
cells  of  the  peritoneum  the  property  of  rapidly  secreting  substances 
harmful  to  the  vibrios  whenever  these  organisms  were  injected  into 
the  peritoneal  cavity.  Metchnikoff  $  soon  showed  that  this  inter- 
pretation is  incorrect.  He  was  able  to  produce  the  granular  change 
of  vibrios  in  vitro  by  mixing  them  with  preventive  serum  plus  the 
peritoneal  exudate  of  a  normal  guinea-pig;  this  exudate  contains 
leucocytes,  but  no  endothelial  cells. 

Pfeiffer  in  his  experiments  failed  to  obtain  a  transformation  on 
mixing  the  vibrios  in  vitro  with  the  serum  of  vaccinated  animals. 
From  our  present  knowledge  it  is  evident  that  the  serum  he 
used  must  have  been  too  old  to  have  retained  its  bacteriolytic 
properties. 

Bordet§  indeed  produced  Pfeiffer's  phenomenon  in  vitro  by 
simply  mixing  the  vibrios  with  fresh  preventive  serum.  He  further 

*  Sur  les  sensibilisatrices  des  se"rum  actifs  centre  les  substances  albuminoi'des. 
Annales  de  1'Institut  Pasteur,  XVI,  1902,  734. 
t  Pfeiffer,  Zeit.  fur  Hyg.  XVIII,  1894. 
}  Metchnikoff,  Annales  de  PInstitut  Pasteur,  June,  1895. 
§  Bordet,  see  p.  66. 

241 


242  STUDIES  IN   IMMUNITY. 

showed  that  the  bacteriolytic  property  of  anticholera  serum  neces- 
sitates the  collaboration  of  two  substances:  one,  the  specific  pre- 
ventive substance,  or  sensitizer  as  he  called  it,  formed  during 
immunization  in  the  animal,  and  able  to  resist  rather  high  temper- 
atures (65-70  degrees) ;  the  second,  Buchner's  alexin,  occurring  in  the 
serum  of  normal  as  well  as  of  immunized  animals,  disappearing 
rapidly  on  standing,  and  easily  destroyed  by  heat  (55  degrees).  The 
alexin  alone  has  little  effect  on  normal  vibrios,  but  becomes  infinitely 
more  active  when  the  vibrios  have  been  acted  on  by  a  specific 
sensitizer. 

This  idea  of  the  duality  of  the  bacteriolytic  substances  was 
established  by  Bordet  in  1895  by  means  of  a  series  of  experiments, 
of  which  the  following  is  the  most  important:  Heating  cholera 
serum  to  55  degrees  deprives  it  of  its  bacteriolytic  properties, 
but,  on  adding  fresh  serum  from  a  normal  animal  to  this  heated 
serum,  the  latter  entirely  recovers  the  energetic  property  it 
possessed  before  being  heated.  In  other  words,  fresh  normal 
serum  "  reactivates "  heated  cholera  serum.  Indeed,  the  two 
substances,  the  collaboration  of  which  is  necessary,  are  present 
in  such  a  mixture. 

The  alexin,  as  Metchnikoff  and  his  school  have  shown,  is  leuco- 
cytic  in  origin;  even  the  destruction  of  vibrios  within  the  leucocytes 
of  normal  animals  is  due  to  this  alexin,  as  is  evident  from  Metch- 
nikoff's  studies  on  phagocytosis. 

Bordet*  later  injected  animals  with  red  blood  cells  of  other 
species  in  the  place  of  vibrios.  The  serum  of  such  animals  ac- 
quires the  property  of  hemolyzing  in  vitro  the  corpuscles  used  for 
injection,  and  this  hemolysis  is  also  due  to  the  collaboration  of 
two  substances :  the  normal  alexin,  destroyed  by  heating  to  55  de- 
grees, and  an  acquired  sensitizer..  What,  then,  are  the  intimate 
reactions  between  the  active  substances  of  the  serum  and  the 
sensitive  cells? 

We  know  that  if  a  suitable  immune  serum,  previously  heated  to 
55  degrees,  is  added  to  its  specific  red  blood  corpuscles,  there 
is  no  hemolysis  because  the  alexin  has  been  destroyed.  Ehrlich  and 
Morgenrothf  showed  that  under  such  conditions  the  corpuscles 

*  See  p.  134. 

t  See  Collected  Studies  on  Immunity.  Ehrlich-Bolduan.  John  Wiley  & 
Sons,  p.  1. 


THE  SENSITIZERS   OF  SERA.  243 

fix  the  sensitizer  of  the  immune  serum,  and  the  decanted  serum  is 
deprived  of  its  specific  properties.  But  blood  cells  fix  substances 
other  than  sensitizers.  If  we  add  corpuscles  to  fresh  immune  serum 
instead  of  to  heated  serum,  they  deprive  the  fluid,  as  Bordet*  proved, 
of  both  sensitizer  and  alexin.  This  same  phenomenon  of  alexin 
absorption  also  occurs  if  a  mixture  of  heated  hemolytic  serum  and 
fresh  normal  serum  is  employed  instead  of  fresh  hemolytic  serum; 
we  know  from  the  experiment  of  Bordet,  to  which  reference  has 
been  made,  that  an  immune  serum  is  completely  regenerated  on  the 
addition  of  fresh  normal  serum  and  becomes  quite  as  active  as  it 
was  before  being  heated.  But  how  may  the  absorption  of  alexin 
in  this  mixture  of  corpuscles,  alexin  and  suitable  sensitizer  be  de- 
tected? The  matter  is  quite  simple:  fresh  sensitized  cells,  say 
cholera  vibrios,  treated  with  heated  cholera  serum,  are  added  to 
the  mixture.  If  there  is  still  alexin  present,  the  sensitized  vibrios 
subsequently  introduced  will  certainly  undergo  granular  transforma- 
tion, but  if  they  remain  intact  it  shows  that  the  alexin  has  disap- 
peared from  the  fluid.  This  experiment  may  be  reversed  by  mixing 
the  alexin  with  the  vibrios  plus  heated  cholera  serum  and  then 
adding  sensitized  corpuscles:  in  this  instance  it  is  the  vibrios  that 
absorb  the  alexin  and  are  destroyed,  and  the  subsequently  added 
corpuscles  remain  intact. 

From  these  experiments  Bordet  drew  the  conclusion  that  the 
alexin  that  is  fixed  by  and  that  destroys  sensitized  blood  cells  is 
identical  with  the  alexin  that  acts  on  sensitized  bacteria.  In  a  given 
serum,  then,  there  would  be  only  one  alexin  attacking  bacteria  and 
corpuscles  indifferently.  Whether  we  agree  with  this  observer  as 
to  the  unity,  or  at  least  the  functional  unity,  of  this  substance, 
or  with  Ehrlich  and  Morgenroth  in  believing  that  a  normal 
serum  contains  a  large  number  of  different  alexins,  each  particularly 
adapted  for  a  given  cell  or  bacterium,  the  fact  remains  that,  in 
presence  of  a  suitable  sensitizer,  a  bacterium  or  a  cell  deprives  fresh 
serum  of  its  alexin  and  renders  it  inactive  either  for  the  same  cell 
or  for  other  sensitized  cells. 

It  is  hardly  necessary  to  state  that  in  these  experiments  the 
alexin,  obtained  as  a  rule  from  fresh  serum  of  the  rabbit  or  guinea- 
pig,  is  not  absorbed  by  the  cells  unless  a  sensitizer  is  present.  In 

*  See  p.  191. 


244  STUDIES  IN   IMMUNITY. 

other  words,  if,  instead  of  the  mixture  of  alexin,  corpuscles  and 
hemolytic  serum  (55  degrees),  a  mixture  is  prepared  in  which  the 
specific  hemolytic  serum  is  replaced  by  normal  serum  from  the 
same  species,  the  fluid  remains  full  of  alexin  and  retains  its  de- 
structive power  for  sensitized  cells.  Normal  sera,  then,  as  a  rule 
are  unable  to  produce  alexin  absorption.  If  normal  serum  con- 
tains sensitizers,  they  are  either  too  little  in  amount  or  too 
inactive  to  cause  a  fixation  of  alexin  as  determined  by  the  method 
described. 

There  are,  however,  certain  exceptions  to  this  general  rule. 
There  are  normal  sera  the  alexin  of  which  may  be  fixed  by  cer- 
tain cells  without  the  presence  of  an  immune  serum.  Malvoz* 
has  recently  shown  that  this  is  the  case  with  dog  serum  mixed 
with  B.  anthracis;  this  serum,  moreover,  in  presence  of  this  organ- 
ism will  cause  the  fixation  of  the  alexins  of  other  sera  (rabbit, 
guinea-pig,  rat) ;  in  other  words,  it  acts  as  if  it  contained  a  true 
sensitizer. 

In  a  similar  way  Bordet  and  I  have  recently  found  that,  in  a 
mixture  of  fresh  normal  dog  serum  and  washed  rabbit  corpuscles, 
hemolysis  occurs  rapidly,  and  a  fixation  of  alexin  takes  place  to 
such  an  extent  that  subsequently  added  sensitized  vibrios  undergo 
no  transformation. 

Such  cases,  however,  are  the  exception.  The  normal  serum  of  the 
majority  of  laboratory  animals  does  not  affect  cells  enough  to 
cause  them  to  fix  alexin  to  an  appreciable  extent ;  immune  sera,  on 
the  contrary,  render  cells  very  avid  of  this  substance.  The  reaction 
of  alexin  fixation  in  general  shows  a  distinction  between  an  immu- 
nized and  a  normal  animal  of  the  same  species.  In  other  words,  it 
demonstrates  the  presence  of  the  specific  sensitizers  produced  by 
vaccination. 

Previous  to  Bordet's  experiments  on  alexin  absorption  a  sensi- 
tizer could  be  defined  simply  as  a  substance  that  renders  a  given 
cell  destructible,  or  at  least  morphologically  alterable,  by  a  dose 
of  alexin  that  normally  has  no  effect  on  it.  But  since  the  sensitizer 
has  not  only  the  property  of  increasing  the  harmful  effect  of  the 
alexin,  but  also  the  property  of  causing  the  cell  it  affects  to  absorb 
*  Annales  de  1'Institut  Pasteur,  August,  1902. 


THE  SENSITIZERS  OF  SERA.  245 

alexin,  no  morphological  change  in  the  specific  cell  is  necessary  to 
prove  the  existence  of  a  sensitizer  in  a  given  serum.  A  disappear- 
ance of  the  alexin  from  the  fluid  is  the  only  essential  criterion.  By 
this  means  Bordet  and  Gengou*  were  able  to  find  sensitizers  in 
the  majority  of  antimicrobial  sera,  as,  for  example,  in  the  sera  of 
animals  injected  with  B.  pestis,  the  bacillus  of  swine  plague,  the 
first  anthrax  vaccine,  B.  typhosus,  B.  proteus  vulgaris,  and  in  the 
serum  of  convalescents  from  typhoid  fever.  If,  for  example,  we  put 
the  same  amounts  of  plague  bacilli  suspended  in  salt  solution,  and 
of  alexin,  in  two  tubes,  and  then  add  to  the  first  a  given  dose  of 
normal  horse  serum  heated  to  55  degrees,  and  to  the  second  the 
same  amount  of  heated  serum  from  a  horse  vaccinated  against  B. 
pestis,  it  will  be  found  that  sensitized  rabbit  corpuscles,  subsequently 
added,  undergo  complete  hemolysis  in  the  first  tube,  but  remain 
intact  in  the  second.  In  other  words,  the  alexin  has  remained  free 
in  the  first  and  disappeared  in  the  second.  Suitable  controls  show 
that  it  is  indeed  the  plague  bacilli  influenced  by  the  preventive 
serum  that  fix  the  alexin. 

The  facts  have  not  been  questioned  so  far  as  we  are  aware; 
Aschoff,!  however,  has  recently  criticised  the  method  employed  by 
Bordet  and  Gengou  on  the  ground  that  the  production  of  hemolysis 
does  not  necessarily  indicate  the  absence  of  a  sensitizer  (probably 
in  a  normal  serum) ;  for,  as  he  says,  "  besides  the  alexins  suitable 
for  bacteriolytic  amboceptors  there  may  be  alexins  fitted  for  hemo- 
lytic  amboceptors. "  This  is  not  the  point  at  issue,  for  B.  &  G.  have 
not  sought  to  establish  that  there  is  no  sensitizer  in  normal  serum, 
but  that  there  is  one  in  immune  serum.  And,  what  is  more,  if 
Aschoff  believes  in  the  existence  of  an  antiplague  sensitizer  in 
normal  horse  serum  as  well  as  in  antiplague  serum,  how  does  he 
explain  that  the  latter  fixes  these  "hemolytic  alexins,"  whereas 
the  former  does  not? 

From  this  summary  it  is  evident  that  hitherto  sensitizers  have 
been  demonstrated  and  studied  only  in  such  sera  as  act  on  definite 
cells.  We  may  go  further  and  consider  whether  the  substance 

*  See  p.  217. 

t  Aschoff :  Ehrlich's  Seitkettentheorie  und  ihre  Anwendung  auf  die  kiinstlichen 
Immunitatsprozesse.  Zeitsch.  f.  allgem.  Physiol.,  1902,  3  Hft  1  ter  Bd.  p.  159. 


246  STUDIES  IN   IMMUNITY. 

injected  must  be  of  definite  morphology  to  give  rise  to  distinct 
sensitizers.  Substances  without  any  cellular  structure  have  already 
been  used  for  injection.  Bordet,* for  example, obtained  a  serum  that 
precipitates  cow's  milk  by  injecting  this  milk  into  rabbits.  Was- 
sermannj  obtained  similar  results  with  several  varieties  of  milk. 
TchistovitchJ  and  Bordet  §  injected  various  foreign  sera  into  rabbits 
and  obtained  corresponding  precipitating  sera.  Mijers||  produced 
sera  that  precipitate  egg  albumin,  sheep-serum  globulin,  ox-serum 
globulin,  and  pepton.  These  experiments  have  been  repeated  and 
extended  by  other  authors  whose  work  need  not  be  mentioned. 

But  hitherto  only  a  precipitating  property  has  been  described 
in  the  serum  of  animals  injected  with  foreign  non-cellular  organic 
substances.  It  seemed  to  us  desirable  to  ascertain  whether  an 
animal  immunized  in  this  way  does  not  produce  substances  similar 
to  antimicrobial  and  hemolytic  sensitizers  as  well.  For  the  demon- 
stration of  these  sensitizers  we  have  used  the  method  of  Bordet 
and  Gengou,  which,  as  already  stated,  is  based  on  the  fixation  of 
the  alexin  by  a  sensitized  cell.  We  have  endeavored  to  determine 
whether  this  fixation  may  be  produced  by  the  serum  of  rabbits  in- 
jected with  such  fluids  as  cow  milk,  hen-egg  albumin,  pure  horse 
fibrinogen,  and  dog  serum  heated  to  55  degrees,  and,  finally,  with 
the  serum  of  guinea-pigs  immunized  against  rabbit  serum  (55  de- 
grees) ;  in  other  words,  we  have  endeavored  to  determine  whether 
the  sera  of  these  treated  animals  contain  a  sensitizer  as  well  as  a 
precipitin  for  the  substances  injected. 

The  serum  of  rabbits  vaccinated  against  cow  milk. — We  injected 
rabbits  with  relatively  large  amounts  of  cow  milk  previously  heated 
to  70  degrees  for  one-half  hour.  They  were  given,  in  successive 
doses,  10,  10,  12  and  12  c.c.  at  intervals  of  7  days.  They  were  bled 
14  days  after  the  last  injection  and  the  separated  serum  heated 
for  a  half  hour  to  56  degrees;  this  serum  we  refer  to  as  Serum 
rabbit  >  milk  56  degrees.  Fresh  normal  rabbit  serum  freed  from 
corpuscles,  after  standing  overnight  at  room  temperature  (16°  C) 
was  uniformly  employed  as  alexin. 

*  Mechanism  of  agglutination,  p.  142. 

t  Deutsche  med.  Wochenschr.,  1899,  No.  80. 

t  Tchistovitch,  Annales  de  1'Institut  Pasteur,  March,  1899. 

§  Bordet,  see  pp.  142,  175. 

||  Centralblatt  fur  Bakt.,  1900,  XXVII. 


THE  SENSITIZERS   OF  SERA.  247 

With  these  materials  the  following  tubes  were  prepared : 

Tube  1  Cow  milk  0.2  c.c. 

S.  rabbit  >  milk,  56  degrees  0.6  c.c. 

Rabbit  alexin  0.1  c.c. 

Tube  2  Cow  milk  0.2  c.c. 

S.  normal  rabbit,  56  degrees  0.6  c.c. 

Rabbit  alexin  0.1  c.c. 

Tube  3  Salt  solution  of  0.75  per  cent  0.2  c.c. 

S.  rabbit  >  milk,  56  degrees  0.6  c.c. 

Rabbit  alexin  0.1  c.c. 

Tube  4  Salt  solution  0.2  c.c. 

S.  normal  rabbit,  56  degrees  0.6  c.c. 

Rabbit  alexin  0.1  c.c. 

Tube  5  Cow  milk  0.2  c.c. 

S.  rabbit  >  milk,  56  degrees  0.6  c.c. 

Salt  solution  0.1  c.c. 

Tube  6  Cow  milk  0.2  c.c. 

S.  normal  rabbit,  56  degrees  0.6  c.c. 

Salt  solution  0.1  c.c. 

Tubes  1  and  2.  In  these  tubes  the  action  of  the  rabbit  >  milk  serum  on  cow 
milk  is  compared  with  that  of  normal  rabbit  serum. 

Tubes  3  and  4.     Show  the  effect  of  the  sera  alone  on  the  alexin. 

Tubes  5  and  6.  Contain  no  alexin  and  show  that  heated  serum  will  not  pro- 
duce hemolysis  without  alexin,  and  act  as  controls  to  those  tubes  in  which  the 
alexin  is  not  affected. 

These  tubes  are  left  5  hours  at  room  temperature,  with  agitation 
from  time  to  time.  To  each  tube  is  then  added  one-thirtieth  of  a 
cubic  centimeter  of  sensitized  hen  corpuscles.*  Following  is  the 
resultant  hemolysis  after  2  hours  at  room  temperature,  which  result 
is  found  to  remain  constant  the  next  day: 

In  tubes  2,  3  and  4  hemolysis  is  complete  and  uniformly  rapid; 
it  is  evident  in  30  minutes  and  has  become  complete  in  1  hour  and 
30  minutes.  Tubes  1,  5  and  6  show  no  hemolysis  even  on  the  fol- 
lowing day.  What  conclusion  is  to  be  drawn?  In  tubes  5  and  6 
there  is  no  hemolysis  because  no  alexin  is  present;  in  tubes  3  and  4 
the  alexin  has  remained  free  when  mixed  with  the  heated  rabbit 
>  milk  serum  or  with  heated  normal  rabbit  serum;  and  in  tube  2  the 
milk  plus  normal  serum  has  caused  no  fixation.  Tube  1  has  acted 
as  tubes  5  and  6  that  had  no  alexin.  The  alexin  added  could  not 
have  been  fixed  by  the  heated  rabbit  >  milk  serum  (tube  3)  nor  by 

*  Defibrinated  hen  blood  is  washed  in  an  excess  of  normal  saline,  centrifugal- 
ized,  the  supernatant  fluid  pipetted  off,  and  the  original  level  of  the  blood  re- 
established. This  washed  blood  is  mixed  with  two  volumes  of  heated  (56  degrees) 
serum  from  a  rabbit  that  has  received  several  injections  of  hen  blood. 


248  STUDIES  IN  IMMUNITY. 

the  milk  (tube  2),  unless  the  latter  had  undergone  some  combina- 
tion with  the  specific  serum  similar  to  that  described  by  Bordet  and 
by  Bordet  and  Gengou  for  bacteria  and  corpuscles  treated  with  their 
appropriate  immune  sera.  We  may  admit,  then,  that  the  serum  of 
rabbits  that  have  been  given  injections  of  cow  milk  contains,  in 
addition  to  the  precipitin,  a  sensitizer  similar  to  those  of  antimi- 
crobial and  hemolytic  sera,  which  gives  cow  milk  the  power  to  fix 
the  alexin  of  normal  serum. 

The  serum  of  rabbits  injected  with  egg  white. — These  animals  were 
given  large  doses  of  egg  white;  at  intervals  of  7  days  they  received 
10,  10,  12  and  10  c.c.  of  egg  white  and  14  days  after  the  last  injec- 
tion they  were  bled.  The  serum  —  designated  serum  rabbit  > egg  — 
was  heated  to  56°  C.  for  30  minutes;  clear  normal  rabbit  serum  was 
used  as  alexin. 

An  experiment  wholly  analogous  to  the  one  with  rabbit  >  milk 
serum,  56  degrees,  was  made  with  this  rabbit  >egg  serum.  In  place 
of  milk,  egg  white  was  used. 

The  results  of  such  an  experiment  are  identical  with  those 
described  for  heated  rabbit  >  milk  serum.  It  is  found  that  alexin 
is  fixed  by  hen-egg  white  in  the  presence  of  rabbit  >  egg  serum 
(56  degrees),  whereas  no  such  fixation  occurs  with  heated  normal 
rabbit  serum  (tube  2)  or  with  egg  white  alone  (tubes  3  and  4). 

The  rabbit  >  egg  serum  also  gives  a  precipitate  with  egg  white 
which  normal  rabbit  serum  fails  to  do. 

The  sera  of  rabbits  injected  with  heated  dog  serum  (56  degrees).  — 
This  serum,  rabbit  >  dog  serum,  56  degrees,  was  obtained  in  a 
manner  similar  to  the  other  sera  described.  The  rabbits  were 
given  at  week  intervals  8,  10  and  12  c.c.  of  dog  serum  that  had  been 
heated  to  56  degrees.  The  treated  rabbits  were  bled  2  weeks  after 
the  last  injection  and  the  separated  serum  was  heated  to  56  degrees 
for  one-half  hour. 

Experiments  similar  to  those  described  for  the  other  active  sera 
were  performed  with  this  serum  active  against  dog  serum;  similar 
controls  were  also  made  and  need  not  again  be  specified.  The 
important  tubes  are  as  follows: 

(a)  Rabbit  alexin,  0.1  of  a  cubic  centimeter,  serum  rabbit  >  dog 
serum,  56  degrees,  0.6  of  a  cubic  centimeter,  dog  serum,  56  degrees, 
0.2  of  a  cubic  centimeter. 


THE  SENSITIZERS  OF  SERA.  249 

(6)  Same  as  last  tube,  with  heated  normal  rabbit  serum  replacing 
the  specific  serum. 

These  mixtures  are  left,  as  usual,  for  5  hours  at  room  tempera- 
ture and  then  one-thirtieth  of  a  cubic  centimeter  of  sensitized  hen 
corpuscles  is  added  to  each  tube. 

The  results  are  wholly  in  harmony  with  those  already  de- 
tailed. The  corpuscles  are  destroyed  by  the  alexin  in  "6";  in 
"a;;,  on  the  contrary,  there  is  no  hemolysis.  The  rabbit  serum 
specific  for  dog  blood  has  fixed  the  alexin  when  dog  serum  is 
present;  in  addition  to  Tchistovitch's  precipitin  there  is  also  a 
sensitizer,  as  in  the  case  of  rabbit  >  milk  serum  and  rabbit  > 
egg  serum. 

The  serum  of  guinea-pigs  treated  with  heated  rabbit  serum.  —  We 
decided  to  study  this  combination  on  account  of  certain  peculi- 
arities that  it  was  known  to  present.  We  know  from  Bordet's 
researches  that,  contrary  to  the  general  rule,  guinea-pigs  injected 
with  rabbit  serum  form  no  precipitin  for  this  serum.  On  account  of 
this  unusual  occurrence  the  question  might  well  arise  as  to  whether 
multiple  injections  instead  of  the  usual  two  injections  might  not 
give  a  different  result.  We  therefore  gave  our  guinea-pigs  six 
successive  injections  of  from  4  to  5  c.c.  of  rabbit  serum.  By  this 
means  we  obtained  a  serum  which,  although  it  produced  no  real 
precipitate,  did  give  rise  to  a  distinct  opalescence  when  rabbit 
serum  was  added. 

As  we  presupposed  from  the  work  of  others,  the  guinea-pig  pro- 
duces only  very  weak  precipitins  to  rabbit  serum.  We  have  also 
tested  for  the  presence  of  a  sensitizer  in  this  "guinea-pig  >  rab- 
bit" serum.  We  found  that  guinea-pig  alexin  is  indeed  fixed  by  a 
mixture  of  rabbit  serum  and  heated  guinea-pig  >  rabbit  serum; 
this  fixation,  however,  is  distinctly  less  than  in  the  other  instances 
considered  and  is  indeed  a  mere  delay  in  hemolysis  rather  than  a 
total  inhibition.  We  consider,  however,  that  a  sensitizing  property 
may  be  claimed  for  this  serum,  although  it  is  as  slight  as  is  the 
precipitating  property. 

The  serum  of  rabbits  treated  with  pure  horse  fibrinogen. — We 
wished  to  determine  whether  injecting  pure  fibrinogen  into  rabbits 
would  lead  to  the  formation  of  sensitizers  for  this  chemically  pure 
substance,  as  we  already  know  that  coagulins  for  the  globulins, 


250  STUDIES  IN   IMMUNITY. 

casein,  etc.,  may  be  produced  in  such  a  way.  We  have  used 
Hammarsten's  method  of  obtaining  horse  fibrinogen.* 

The  rabbits  were  given  at  intervals  of  7  days  10,  12,  12  and 
15  c.c.  of  this  solution  of  fibrinogen  and  were  bled  2  weeks  after 
the  last  injection.  The  serum,  designated  rabbit  >  fibrinogen 
serum,  56  degrees,  was  tested  with  the  pure  fibrinogen.  An  abun- 
dant precipitum  is  formed  at  once  on  mixing;  no  such  precipitate 
is  formed  with  normal  rabbit  serum.  We  were  further  able  to 
determine  by  the  method  described  that  the  rabbit  >  fibrinogen 
serum  fixes  rabbit  alexin  in  the  presence  of  fibrinogen.  In  other 
words,  this  immune  serum  contains  a  sensitizer  for  fibrinogen. 

From  these  facts  we  may  conclude  that  rabbits  injected  with  such 
substances  as  milk,  egg  white,  fibrinogen,  and  serum,  form  both 
precipitins  and  sensitizers  for  the  respective  substances.  The  pro- 
duction of  sensitizers,  then,  is  not  dependent  on  the  morphology  of  the 
substance  injected  and  the  animal  body  mil  react  as  well  against  amor- 
phous material  as  against  substances  of  definite  histological  structure. 

Just  as  the  sensitizers  of  antimicrobial  and  hemolytic  sera  fix 
the  alexin  of  normal  serum  in  presence  of  the  corresponding  antigen, 
so  do  the  sensitizers  of  sera  active  against  amorphous  organic  pro- 
ducts fix  the  alexin  in  presence  of  the  substance  in  question. 

The  majority  of  the  immune  sera  we  have  worked  with  act  on 
complex  mixtures  like  serum,  milk  and  egg  albumin.  It  would  be 
of  interest  to  determine  whether  the  sensitizer  acts  on  the  complex 
as  a  whole  or  more  particularly  on  one  or  several  components  of  it. 

Several  writers  have  considered  this  question  in  respect  to  coagu- 
lins  and  with  somewhat  divergent  results.  It  may  be  considered 
proved,  it  seems  to  us,  that  the  coagulin  affects  the  globulins 

*  Oxalated  horse  plasma  (1  to  1000)  is  centrifugal ized  and  mixed  with  an  equal 
volume  of  30  per  cent  solution  of  sodium  chloride.  The  precipitate  is  redissolved 
in  8  per  cent  salt  solution  and  again  precipitated  with  the  30  per  cent  solution; 
after  three  or  four  successive  precipitations  the  fibrinogen  is  redissolved  in  sterile 
distilled  water,  as  is  possible  owing  to  the  sodium  chloride  carried  down  by  the 
precipitate.  To  test  its  purity  the  fibrinogen  is  mixed  on  the  one  hand  with  cal- 
cium chloride  and  on  the  other  with  fresh  blood  serum.  In  the  second  mixture 
coagulation  occurs,  proving  there  is  fibrinogen  present  in  solution.  No  coagu- 
lation takes  place  in  the  first  tube,  showing  that  the  proferment  is  absent. 
We  further  determined  by  heat  that  no  other  albumins  or  globulins  were 
present. 


THE  SENSITIZERS   OF  SERA.  251 

(Nolf,*  Mijers,f  F.  Hamburger,  J  Van  Steenberghe,§  Leblano||).  It 
is  difficult  to  determine  the  effect  on  the  albumins ;  F.  Hamburger 
claims  to  have  obtained  a  coagulin  for  lactalbumin,  Mijers  one  for 
egg  albumin,  and  Leblanc  one  for  serum  albumin;  but  Nolf,  on  the 
other  hand,  could  obtain  no  coagulin  for  serum  albumin. 

We  have  made  a  few  observations  along  this  line,  restricting 
ourselves  to  the  action  of  rabbit  >  dog  serum  and  rabbit  >  milk 
serum  on  the  separable  substances  of  their  respective  antigens. 

The  effect  of  rabbit  >  dog  serum  on  the  globulins  and  the  albumins 
of  dog  serum.  —  We  separated  the  globulins  from  dog  serum  by 
saturation  with  magnesium  sulphate.  After  redissolving  in  dis- 
tilled water  the  globulins  were  again  precipitated  by  sodium  chlo- 
ride; this  precipitation  was  repeated  twice  and  the  final  globulins 
were  dissolved  in  a  volume  of  distilled  water  to  equal  their  original 
serum  volume.  Solution  is  made  possible  by  the  NaCl  taken  down 
by  the  globulins. 

The  albumin  is  obtained  from  the  fluid  of  the  original  precipita- 
tion with  magnesium  sulphate  by  adding  acetic  acid,  and  is  redis- 
solved  in  normal  salt  solution ;  the  fluid  is  then  neutralized  to  litmus 
by  the  addition  of  a  few  drops  of  2  per  cent  NaOH. 

With  these  two  products,  two  series  of  tubes  are  prepared  con- 
taining mixtures  of  normal  rabbit  serum  or  specific  rabbit  serum 
with  and  without  rabbit  alexin.  In  the  mixtures  without  alexin 
the  sensitized  hen  corpuscles  added  5  hours  later  remain  intact;  in 
other  words,  the  solutions  of  globulins  and  albumins  employed  were 
not  in  themselves  hemolytic.  In  the  mixture  of  normal  rabbit 
serum  and  alexin  hemolysis  occurred;  there  is  no  hemolysis,  however, 
in  the  mixtures  containing  rabbit  >  dog  serum. 

It  would  seem,  then,  that  rabbit  >  dog  serum  will  fix  rabbit  alexin 
with  either  the  globulin  or  the  albumin  of  dog  serum.  We  do 
not  wish,  however,  to  attach  too  much  significance  to  this  experi- 
ment, which  we  were  unfortunately  unable  to  repeat  owing  to  lack 
of  material. 

The  effect  of  rabbit  >  milk  serum  on  casein,  lactoglobulin  and 

*  Nolf,  Annales  de  Tlnstitut  Pasteur,  1900. 

f  Mijers,  loc.  cit. 

%  F.  Hamburger,  Wien.  klin.  Wochens.,  1901,  p.  1202. 

§  Van  Steenberghe,  Annales  de  1'Institut  Pasteur,  1901. 

II  Leblanc,  La  Cellule,  t.  XVIII,  2nd  fascic. 


252  STUDIES  IN   IMMUNITY. 

lactalbumin  from  cow  milk.  —  These  experiments  with  the  various 
milk  derivatives  have  been  frequently  repeated  and  always  with 
the  same  result. 

We  obtained  milk  casein  by  the  well-known  method  of  diluting 
with  three  volumes  of  water  and  precipitating  with  0.1  to  0.2  per 
cent  acetic  acid.  The  resultant  precipitate  is  redissolved  with 
ammonia,  1  to  200,  and  reprecipitated  twice  with  acetic  acid. 

The  casein  purified  in  this  manner  is  dissolved  in  water,  rendered 
alkaline  with  ammonia  and  made  equal  in  amount  to  the  original 
volume  of  the  milk;  neutralization  is  then  made  with  phosphoric 
acid. 

The  lactoglobulin  and  the  lactalbumin  were  obtained  from  whey 
by  the  same  method  employed  for  dog  serum.  They  were  all 
finally  redissolved  in  a  much  smaller  amount  of  fluid  than  the 
original  whey  volume.  We  know  from  Bordet's  studies  that  in 
order  to  produce  complete  fixation  of  alexin  by  bacteria  and  spe- 
cific serum  that  a  considerable  number  of  bacteria  are  necessary. 
Since  lactoglobulin  and  lactalbumin  are  present  in  such  small 
amounts  in  milk  we  should  not  expect  them,  in  absence  of  casein, 
which  forms  the  bulk  of  organic  substances,  to  fix  all  the  alexin  in 
the  presence  of  rabbit  >  milk  serum.  • 

With  these  substances  prepared  in  this  way  three  identical  series 
of  tubes  are  prepared  corresponding  to  those  in  the  experiment 
with  rabbit  >  milk  serum  and  whole  milk.  In  addition  to  a  fourth 
control  series  of  milk  and  rabbit  >  milk  serum  there  are  the  three 
other  series  containing  casein,  lactoglobulin,  and  lactalbumin 
respectively  in  place  of  whole  milk. 

In  the  tubes  of  each  series  that  contain  normal  rabbit  serum,  56 
degrees  plus  each  of  the  substances  in  turn,  hemolysis  is  complete; 
the  alexin  is  not  fixed  by  any  of  these  substances  without  rabbit  > 
milk  serum.  But  in  the  mixtures  of  milk  or  milk  derivatives  and 
specific  serum,  hemolysis  occurs  only  in  the  mixture  containing 
lactalbumin,  and  is  lacking  in  the  tubes  containing  casein  and 
lactoglobulin  as  well  as  in  the  one  with  whole  milk.  In  other  words, 
alexin  is  fixed  by  rabbit  >  milk  serum  with  both  casein  and  lacto- 
globulin; it  is  not  fixed,  however,  with  lactalbumin.  We  may  add 
that  the  precipitating  property  of  the  serum  parallels  the  sensitizing 
property;  we  obtain  a  distinct  precipitate  by  adding  casein  or  lacto- 
globulin to  the  specific  serum,  whereas  the  addition  of  lactalbumin 


THE  SENSITIZERS   OF  SERA.  253 

produces  no  such  effect.    We  do  not  agree,  therefore,  with  F.  Ham- 
burger on  this  point. 

One  of  the  principal  properties  of  antimicrobial  and  of  hemolytic 
sensitizers  is  their  specificity.  As  a  general  rule  immune  sera  are 
active  only  against  those  cells  which  by  injection  have  given  rise 
to  them.  There  is,  we  believe,  no  exception  to  this  rule,  although 
Ehrlich  and  Morgenroth  have  noted  that  the  serum  of  rabbits 
treated  with  ox  blood  sensitizes  not  only  ox  corpuscles,  but  also 
goat  corpuscles. 

On  the  other  hand  there  are  numerous  exceptions  to  this  rule 
among  the  coagulins.  Wassermann  and  Schiitze,*  Stern, t  and 
Nuttall  J  in  particular  have  shown  that  the  serum  of  animals  in- 
jected with  human  serum  precipitates  not  only  this  serum,  but  also 
the  serum  of  anthropoid  apes.  Grunbaum,  §  by  injecting  rabbits 
with  the  sera  from  three  different  species  of  monkeys,  obtained  sera 
affecting  the  serum  of  these  three  species  and  also  of  man.  Linos- 
sier  and  Lemoine  ||  found  that  the  serum  of  animals  given  ox  serum 
would  precipitate  sera  of  other  animal  species.  And  Moro  If  has 
recently  shown  that  a  serum  that  precipitates  cow  milk  will  also 
precipitate  goat  milk. 

It  seemed  to  us  well  to  determine  to  what  extent  our  sera  active  ** 
against  albuminous  substances  were  endowed  with  specificity,  par- 
ticularly as  regards  their  sensitizing  property. 

Rabbit  serum  active  against  milk.  —  This  serum  from  rabbits 
that  had  been  given  injections  of  cow  milk  was  heated  to  56  degrees 
and  added  to  different  milks:  cow  milk,  ewe  milk,  goat  milk,  mare 
milk  and  human  milk. 

With  each  milk,  mixtures  were  made  with  normal  rabbit  serum, 
56  degrees,  and  with  specific  serum,  56  degrees. 

Tube  1  Milk  (e.g.,  cow)  0.2  c.c. 

Serum  rabbit  >  milk,  56  degrees  0.6  c.c. 

Alexin  (rabbit)  0.1  c.c. 

Tube  2  Milk  0.2  c.c. 

Serum  normal  rabbit,  56  degrees  0.6  c.c. 

Alexin  0.1  c.c. 

*  Wassermann  and  Schutze.     Berlin  klin.  Wochen.,  XXXVIII,  1901,  187. 

t  Stern,  Deutsche  med.  Wochens.,  1901,  p.  135. 

t  Nuttall,  The  Journal  of  Hyg.,  July,  1901,  3. 

§  Grunbaum,  The  Lancet,  January  18,  1902. 

II  Linossier  and  Lemoine,  Comptes  rend.  Soc.  de  Biol.,  1902   85. 

H  Moro,  Wiener  klin.  Wochsch.,  1902,  121. 


254  STUDIES   IN  IMMUNITY. 

In  addition  to  this  series,  comprising  two  tubes  for  each  milk 
tested,  other  tubes  with  decreasing  doses  of  active  and  normal 
serum,  e.g.,  0.4,  0.2  and  0.1  of  a  cubic  centimeter,  but  all  containing 
the  same  amount  of  alexin  and  milk,  were  prepared. 

In  none  of  the  tubes  containing  normal  rabbit  serum,  56  degrees, 
do  the  various  milks  fix  alexin;  in  all  tubes  containing  rabbit  >  milk 
serum,  on  the  contrary,  an  alexin  absorption  occurs.  There  is  no 
evidence  of  specificity  with  this  serum  in  a  dose  of  0.6  of  a  cubic 
centimeter  of  serum,  0.1  of  a  cubic  centimeter  of  alexin,  and  0.2 
of  a  cubic  centimeter  of  each  milk  in  turn.  Nor  does  decreasing 
the  active  serum  to  0.1  of  a  cubic  centimeter  show  any  difference 
between  the  cow,  ewe  and  goat  milk.  Human  milk,  however,  was 
less  perfectly  sensitized  by  the  serum,  even  in  a  dose  of  0.6  of  a 
cubic  centimeter  of  serum  to  0.2  of  a  cubic  centimeter  of  milk; 
fixation  was  not  quite  complete,  and  is  almost  nil  with  0.2  of  a 
cubic  centimeter  of  serum. 

Mare  milk  seems  to  lie  between  the  first  group  of  milks  and 
human  milk ;  a  total  fixation  occurs  with  0.6  of  a  cubic  centimeter 
of  serum,  0.4  of  a  cubic  centimeter  or  even  with  0.2  of  a  cubic  cen- 
timeter, but  only  an  imperfect  fixation  with  0.1  of  a  cubic  centimeter. 

To  sum  up ;  the  sensitizer  in  our  rabbit  >  milk  serum  is  not  specific. 
It  acts  energetically  on  milks  from  various  allied  animal  species  (cow, 
ewe,  goat)  and  somewhat  less  distinctly  on  other  milks  (human  and 
mare). 

Rabbit  >  egg  serum,  56  degrees.  —  Similar  experiments  were  per- 
formed with  the  sensitizer  of  rabbit  >  egg  serum.  The  serum  was 
obtained  by  injecting  hen-egg  white  and  was  tested  with  egg  white 
from  the  hen,  the  pigeon,  the  turkey  and  the  duck.  On  the  first 
experiments  we  used  uniformly  0.2  of  a  cubic  centimeter  of  serum; 
an  intense  precipitate  was  found  with  all  the  albumins,  and  the 
alexin  was  fixed  in  all  cases  so  that  no  hemolysis  of  sensitized  hen 
corpuscles  occurred.  Hemolysis  was  complete  in  controls  with 
normal  rabbit  serum. 

We  have  not  tried  to  establish  a  difference  between  the  various 
egg  whites  by  using  varying  amounts  of  active  serum,  as  in  the 
rabbit  >  milk  experiments;  such  an  experiment  was  tried,  however, 
between  hen  and  pigeon  albumin.  With  both  species  fixation  is 
complete  with  0.6  or  even  0.5  of  a  cubic  centimeter  of  active  serum. 


THE  SENSITIZERS   OF  SERA.  255 

With  0.4  of  a  cubic  centimeter  it  is  nearly  complete  and  with  0.2 
of  a  cubic  centimeter  very  slight.  There  is  no  differentiation,  then, 
between  pigeon-egg  white  and  hen-egg  white  by  sensitization  with 
rabbit  >  hen-egg  white  serum. 

In  brief,  we  have  found  no  specificity  either  as  regards  precipitin 
with  or  sensitizer  with  rabbit  >  egg  serum. 

Rabbit  >  fibrinogen  serum,  56  degrees. — We  have  found  no  more 
evidence  of  specificity  as  regards  precipitation  and  sensitization 
with  this  serum  than  with  the  two  preceding  sera.  The  serum  was 
obtained  by  injecting  large  amounts  of  pure  fibrinogen  from  the 
horse  and  was  tested  with  fibrinogens  from  the  horse,  the  ox  and 
the  dog,  all  prepared  in  exactly  the  same  way.  An  abundant  and 
immediate  precipitate  occurred  with  each  fibrinogen;  there  was 
apparently  no  difference  in  amount  even  when  the  active  serum 
was  decreased  to  0.1  of  a  cubic  centimeter,  with  0.2  of  a  cubic  cen- 
timeter of  fibrinogen  and  0.1  of  a  cubic  centimeter  of  rabbit  alexin. 

The  same  results  were  obtained  as  regards  sensitization  of  the 
three  fibrinogens.  Whatever  the  dose  of  active  serum  employed 
(3,  2,  1  or  one-half  volume  to  1  volume  of  fibrinogen  solution,  and 
one-half  volume  of  rabbit  alexin),  a  complete  fixation  of  alexin 
occurred  with  each  fibrinogen  with  the  largest  dose  of  serum,  and 
progressively  less  as  the  serum  was  decreased. 

Rabbit  serum  active  for  dog  serum,  56  degrees. — On  account  of  the 
practical  value  of  the  serum  precipitation,  first  demonstrated  by 
Tchistovitch,  we  have  studied  the  specificity  of  our  rabbit  >  dog 
serum. 

Varying  amounts  of  serum  (0.6,  0.4,  0.2  and  0.1  of  a  cubic  cen- 
timeter) were  mixed  with  0.2  of  a  cubic  centimeter  of  various  sera 
heated  to  56  degrees  plus  0.1  of  a  cubic  centimeter  of  rabbit  alexin. 

We  tested  in  this  way  dog,  horse,  ox  and  guinea-pig  serum. 

With  dog  serum  the  precipitate  formed  with  0.6  and  0.4  of  a 
cubic  centimeter  of  specific  serum  is  very  distinct;  it  is  less  with 
0.2  of  a  cubic  centimeter  and  no  longer  visible  with  0.1  of  a  cubic 
centimeter.  With  the  other  sera  no  precipitate  occurred.  Simi- 
larly, the  alexin  was  fixed  only  with  dog  serum  and  more  or  less 
completely  according  to  the  amount  of  specific  serum  employed. 

The  active  serum,  however,  produced  no  alexin  fixation  with  any 
of  the  other  sera  in  any  dose. 


256  STUDIES  IN   IMMUNITY. 

The  specificity  of  the  sensitizer  in  this  instance  was  as  distinct  as 
that  of  the  precipitin,  and  is  analogous  to  the  specificity  of  antimicrobial 
and  hemolytic  sensitizers. 

There  remains  to  describe  certain  experiments  that  we  have 
made,  suggested  by  the  lack  o£  specificity  in  many  of  our  sera.  We 
have  endeavored  to  determine  whether  these  sera  were  active  only 
against  the  particular  substances  used  in  immunization  and  were 
without  effect  on  other  substances  from  the  same  animal  species. 
We  can  simply  add  a  few  examples  to  those  described  by  other 
authors.  Leclainche  and  Vallee,*  Mertens,f  Dieudonne,J  Zuelzer  § 
and  Schiitze  ||  found  that  antihuman  sera  produced  by  injecting 
human  blood  would  give  precipitates  with  urine  and  pleural  exudates 
from  human  beings;  conversely,  the  injection  of  exudates  and  trans- 
udates  gives  rise  to  a  serum  that  precipitates  human  blood. 

Schutze^f  by  injecting  powdered  human  muscle,  obtained  a  serum 
that  sensitized  human  red  blood  cells.  F.  Hamburger**  has  shown 
that  sera  that  precipitate  cow  lactoglobulin  and  lactalbumin  will 
also-  precipitate  bovine  blood  serum. 

We  have  considered  whether  hen-egg  white  might  not  be  pre- 
cipitated and  sensitized  by  a  serum  other  than  the  rabbit  >  egg 
serum.  In  the  following  experiment  we  determined  the  effect 
of  the  serum  of  a  rabbit  treated  with  defibrinated  hen  blood,  and 
very  active  against  the  corpuscles  in  question,  on  hen-egg  white: 

Tube  1  Hen-egg  white  0.2  c.c. 

S.  rabbit  >  hen  blood,  56  degrees  0.6  c.c. 

Rabbit  alexin  0.1  c.c. 

Tube  2  Hen-egg  white  0.2  c.c. 

S.  rabbit  >  egg,  56  degrees  0.6  c.c. 

Rabbit  alexin  0.1  c.c. 

Tube  3  Hen-egg  white  0.2  c.c. 

S.  normal  rabbit,  56  degrees  0.6  c.c. 

Rabbit  alexin  0.1  c.c. 

After  5  hours  contact,  one-thirtieth  of  a  cubic  centimeter  of 
sensitized  hen  blood  was  added  to  each  tube. 

*  Leclainche  and  Valtee,  La  Semaine  mddicale,  1901,  No.  4. 

t  Mertens,  Deutsche  med.  Woch.,  1901,  No.  1. 

J  Dieudonne*,  Munch  med.  Woch.,  1901,  No.  4. 

§  Zuelzer,  Deutsche  med.  Woch.,  1901,  p.  219. 

II  Schutze,  Zeit.  fur  Hyg.,  XXXVI,  1901,  459. 

IT  Schutze,  Deutsche  med.  Woch.,  XXXVIII,  1901,  487. 

**  F.  Hamburger,  Wiener  klin.  Woch.,  1901,  1202. 


THE  SENSITIZERS   OF  SERA.  257 

In  the  first  two  tubes  a  marked  cloudiness  appeared,  which  was 
lacking  in  tube  3.  In  these  two  first  tubes  the  fixation  of  alexin 
also  was  complete,  but  there  was  none  in  tube  3.  The  serum  of 
rabbits  injected  with  defibrinated  hen  blood  is  capable  of  precipitating 
and  of  sensitizing  hen-egg  white  as  well  as  rabbit  >  egg  serum. 

Is  the  converse  true?  That  is  to  say,  will  rabbit  >  egg  serum 
agglutinate  and  sensitize  hen  blood  corpuscles?  The  experiment 
to  answer  this  question  contains  three  tubes: 

Tube  1  Washed  hen  corpuscles  0.1  c.c. 

S.  normal  rabbit,  56  degrees  0.3  c.c. 

Rabbit  alexin  0.2  c.c. 

Tube  2  Washed  hen  corpuscles  0.1  c.c. 

S.  rabbit  >  egg,  56  degrees  0.3  c.c. 

Rabbit  alexin  0.2  c.c. 

Tube  3  Washed  hen  corpuscles  0.1  c.c. 

S.  rabbit  >  hen  blood,  56  degrees  0.3  c.c. 

Rabbit  alexin  0.2  c.c. 

There  is  no  hemolysis  in  tube  1  and  rapid  hemolysis  in  tube  3; 
in  2,  hemolysis  occurs,  but  slowly,  and  apparently  is  not  accom- 
panied by  agglutination. 

We  then  determined  whether  this  rabbit  >  egg  serum  would  act  on 
normal  hen  serum ;  there  was  no  effect.  In  a  similar  manner  rabbit 
>  milk  serum  has  no  effect  on  cow  serum.  In  short,  it  is  evident 
that  if  an  immune  serum  is  tested  on  elements  other  than  those  used 
for  immunization,  but  from  the  same  animal  species,  the  results 
vary  and  cannot  be  prognosticated  in  an  untried  combination. 
Although  rabbit  >  milk  serum  has  no  effect  on  cow  serum  and 
rabbit  >  egg  serum  none  on  hen  serum,  we  find  that  rabbit  > 
egg  serum  will  sensitize  hen  corpuscles  to  a  certain  extent,  and 
that  rabbit  >  hen  blood  serum  is  very  active  against  hen-egg  white. 

CONCLUSIONS 

These  experiments  would,  we  believe,  lead  to  the  conclusions: 
1.  That  in  the  sera  obtained  by  injecting  rabbits  with  large 
doses  of  cow  milk,  egg  white,  pure  horse  fibrinogen,  or  heated  dog 
serum  there  are,  in  addition  to  the  precipitins  of  Bordet  and 
Tchistovitch,  substances  analogous  to  the  sensitizers  described  by 
Bordet  in  bacteriolytic  and  hemolytic  sera,  and  later  found  in  the 
majority  of  antimicrobial  sera.  The  same  is  true  in  respect  to  the 


258  STUDIES  IN   IMMUNITY. 

sera  of  guinea-pigs  injected  with  rabbit  blood,  although  in  this 
case  the  sensitizer  appears  less  powerful. 

2.  The  sensitizers  studied  by  Bordet  cause  blood  cells  or  bac- 
teria to  fix  alexin.    The  sensitizers  we  have  just  described  produce 
the  same  phenomenon  and  differ  only  in  being  directed  against  non- 
differentiated  substances.     We  have  dealt  with  amorphous  chemical 
substances  and  hot  with  morphologically  defined  elements. 

3.  The  sensitizer  in   the  serum  of   rabbits  injected  with  dog 
blood  would  seem  to  act  both  on  the  globulin  and  the  albumin  of 
dog  serum;  the  sensitizer  in  the  serum  of  rabbits  injected  with  cow 
milk  acts  on  the  casein  and  the  lactoglobulin,  but  not  on  the  lact- 
albumin. 

4.  It  is  known  that  the  sensitizers  of  antimicrobial  and  of  hemo- 
lytic  sera  generally  have  the  character  of  marked  specificity.    We 
have  found  this  to  be  true  also  of  the  serum  of  rabbits  active  against 
dog  serum.    On  the  contrary,  the  sensitizers  of  most  of  the  sera 
we  have  studied  have  slight  specificity  or  none  at  all.    Such  sera, 
notably,  are  those  from  animals  injected  with  milk,  egg  white  and 
fibrinogen.    It  is  true  that  these  substances — the  albuminoids  of 
milk,  fibrinogen  and  egg  white  —  show  an  almost  identical  con- 
stitution in  various   animal   species,  at  least  sufficient  to  render 
them  all  susceptible  to  a  given  active  serum  irrespective  of  their 
origin. 

5.  The  sensitizers  of  immune  sera  may  often  act  on  substances 
other  than  those  used  for  immunization,  but  from  the  same  animal 
species.     For  example,  the  serum  of  an  animal  injected  with  hen 
blood  acts  on  hen-egg  white.  There  is  no  general  rule  in  this  respect, 
however,  as  individual  cases  vary. 


XIII.    ON  THE  MODE   OF  ACTION  OF  ANTITOXINS 
ON  TOXINS.* 

BY  DR.   JULES   BORDET. 

The  majority  of  scientists  who  have  studied  antitoxins  believe 
that  they  modify  the  poison  for  which  they  are  the  antidote.  In 
other  words,  antitoxin  protects  the  animal,  not  by  rendering  it  more 
resistant,  but  by  reacting  with  the  toxin  and  destroying  its  harm- 
ful properties. 

This  conclusion  has  been  drawn  from  numerous  important  re- 
searches, among  which  may  be  mentioned  those  of  Martin  and 
Cherry.  Its  accuracy  became  very  evident  as  soon  as  we  were 
able,  thanks  to  Ehrlich,  to  eliminate  experiments  on  the  living 
animal,  with  the  accompanying  uncertainty  of  individual  varia- 
tion, and  replace  them  by  cells  that  are  susceptible  to  the  poison. 
Thereafter  we  were  able  to  study  the  effect  of  antitoxin  on  toxin 
in  vitro,  as  the  sensitive  cell  is  changed  when  toxin  is  present,  but 
remains  intact  if  this  poison  has  been  neutralized  by  a  suitable  dose 
of  antitoxin. 

The  conception  that  antitoxin  not  only  modifies  toxin,  but  that 
it  also  unites  with  it,  is  in  perfect  harmony  with  the  data  we  have 
concerning  the  other  active  substances  of  sera,  and  may  therefore 
be  accepted  with  relative  certainty.  The  action  resembles  closely 
the  effect  produced  by  agglutinins,  sensitizers  and  alexin  on  sus- 
ceptible cells;  in  a  similar  way  the  precipitins,  which  are  strikingly 
analogous  to  the  agglutinins,  agree  to  a  certain  extent  with  true 
antitoxins  in  that  they  unite  with  non-differentiated  chemical 
substances. 

Since  we  may  regard  it  as  proven  that  antitoxin  destroys  the 
harmful  properties  of  toxin  by  direct  combination,  an  attempt  may 
be  made  to  determine  the  intimate  reaction  between  these  two 

*  Sur  le  mode  d'action  des  antitoxines  sur  les  toxines.  Annales  de  1'Institut 
Pasteur,  XVII,  1903,  161. 

259 


260  STUDIES  IN  IMMUNITY. 

substances.  Many  investigators  have  endeavored  to  solve  this 
problem,  but,  of  necessity,  with  only  partial  success.  As  the  molec- 
ular constitution  of  these  substances  is  a  mystery  and  their  chemi- 
cal nature  unknown,  their  interaction  naturally  could  not  be  so 
exactly  and  clearly  demonstrated  as  is  possible  in  dealing  with  the 
well-known  substances  with  which  chemists  work. 

We  must  be  content,  then,  for  the  present  with  outlining  the 
general  characters  of  the  reaction,  in  describing  its  appearance  and 
in  endeavoring  to  determine  the  laws  to  which  it  is  subject.  The 
first  question  to  answer  obviously  is :  does  antitoxin  unite  with  toxin 
in  definite  proportions,  as  does  a  monobasic  acid  with  an  alkali, 
in  which  case  the  neutralized  product  has  a  fixed  and  invariable 
composition;  or  does  one  of  the  substances  unite  with  the  other 
in  varying  amounts?  If  this  second  supposition  is  correct,  it  is 
evident  that  the  combination  formed  by  union  of  the  two  substances 
will  not  always  be  the  same  or  endowed  with  the  same  characteris- 
tics, but  will  vary  in  composition  according  to  the  relative  propor- 
tions of  the  two  reacting  substances.  For  instance,  a  mixture  of 
equal  volumes  of  toxin  and  antitoxin  would  produce  a  different 
substance  than  a  mixture  of  one  part  of  toxin  with  two  of  antitoxin. 
The  resulting  substance  would  vary  with  the  respective  doses  of 
the  two  components  employed :  all  would  contain  the  same  elements, 
toxin  and  antitoxin,  but  would  differ  in  that  one  substance  would 
be  more  or  less  saturated  by  the  other.  According  to  this  hypothe- 
sis, the  reaction  of  toxin  and  antitoxin  would  resemble,  at  least  in 
regard  to  the  variation  of  proportions,  the  reaction  of  iodine  on 
starch.  Starch,  as  we  know,  absorbs  varying  amounts  of  iodine 
and  correspondingly  varies  in  the  intensity  of  its  blue  color;  for 
this  reason  chemists  regard  this  reaction  as  belonging  to  the  phe- 
nomena of  dyeing.  Dyed  substances  take  widely  varying  amounts 
of  the  dye. 

Let  us  consider  for  a  moment  this  comparison  with  dyeing  phe- 
nomena, a  comparison  that  we  have  previously  made  use  of,  and 
it  would  seem  inadvisedly,  since  it  has  led  many  who  have  read 
our  preceding  articles  to  a  thorough  misconception  of  them. 

As  we  have  determined,*  the  maximal  amount  of  red  blood  cells 
that  a  given  dose  of  hemolytic  serum  can  destroy  varies  in  accord- 

*  See  p.  194. 


ACTION  OF  ANTITOXINS  ON  TOXINS.  261 

ance  with  the  manner  in  which  the  blood  is  added  to  the  serum. 
If,  for  example,  we  add  a  given  amount  of  blood  in  a  single  dose  to, 
say,  1  c.c.  of  serum,  this  amount,  A,  may  be  relatively  considerable 
and  yet  be  entirely  hemolyzed.  But  if  this  amount  A  is  divided 
into  several  fractions  added  one  after  another  at  sufficiently 
spaced  intervals  to  the  same  amount  of  the  active  serum,  only  a 

few  of  the  corpuscles,  say   ->  will  be  destroyed.     It  would  seem 

as  if  the  first  doses  of  corpuscles  had  taken  up  all  the  hemolytic 
substances,  and  so  deprived  the  subsequently  added  corpuscles  of 
their  share. 

A  blood  corpuscle,  then,  absorbs  varying  amounts  of  active  sub- 
stances, the  maximal  dose  that  can  be  fixed  being  distinctly  greater 
than  the  amount  necessary  to  produce  complete  solution.*  With 
this  explanation  we  may  offer,  as  a  preliminary  hypothesis,  that 
the  absorption  of  the  active  principles  of  serum  by  the  fixing  portion 
of  blood  cells  does  not  follow  the  law  of  fixed  proportions,  but 
resembles,  rather,  the  absorption  of  dyes  by  substances  that  take 
them,  which  absorption  varies  considerably  in  amount.  Con- 
sequently we  should  expect  the  absorbing  energy  to  vary  to  a 
large  extent,  depending  on  the  conditions  of  the  experiment  (con- 
centration of  the  substance  considered,  length  of  contact,  establish- 

*  As  we  know,  hemolysis  depends  on  the  collaboration  of  the  alexin  and  the 
sensitizer.  To  which  of  these  two  substances  is  this  result  due?  In  other  words, 
which  one  is  absorbed  in  variable  doses  by  the  red  blood  cell?  Both  substances 
have  this  property,  but  the  alexin  shows  it  the  more  distinctly;  and  to  the  alexin, 
then,  is  due  the  greater  part  in  the  phenomenon.  To  a  tube  containing  0.5  of  a 
cubic  centimeter  of  alexin  (fresh  guinea-pig  serum)  is  added  0.3  of  a  cubic  cen- 
timeter of  well-washed  rabbit  blood,  and  immediately  afterward  0.9  of  a  cubic 
centimeter  of  sensitizer  (guinea-pig  >  rabbit  serum,  55  degrees),  hemolysis  follows 
rapidly.  Three  hours  later  0.1  of  a  cubic  centimeter  more  of  blood  and  0.3  of  a 
cubic  centimeter  of  sensitizer  is  added,  and  1  hour  later  0.1  of  a  cubic  centimeter 
more  of  blood  and  0.3  of  a  cubic  centimeter  of  sensitizer.  The  last  corpuscles 
remain  intact.  We  may  then  prepare  a  mixture  containing  the  same  total 
amount  of  each  substance  (0.5  c.c.  of  alexin,  0.5  c.c.  of  blood  and  1.5  c.c.  of 
sensitizer),  but  mix  them  together  at  once.  Complete  hemolysis  occurs.  The 
intact  corpuscles  in  the  first  mixture  are  well  sensitized,  but  lack  alexin.  This  is 
shown  by  the  fact  that  the  subsequent  addition  of  alexin  produces  complete 
hemolysis.  We  may  conclude,  then,  that  the  stromata  of  the  first  hemolyzed  cor- 
puscles are  loaded  with  alexin  that  they  refuse  to  yield  to  other  sensitized  corpus- 
cles. The  stroma-alexin  complex  is  stable  and  does  not  break  up.  We  shall  later 
return  to  this  fact  in  considering  one  of  Morgenroth's  experiments. 


262  STUDIES  IN  IMMUNITY. 

ing  of  equilibrium  between  the  dose  of  active  substance  absorbed 
and  that  which  remains  free,  and  so  forth),  and  this  idea  we  have 
endeavored  to  prove  experimentally. 

We  may  add  that  this  interpretation  has  appealed  to  various 
observers  who  have  performed  similar  experiments ;  similar  and  even 
more  demonstrative  results  have  been  obtained,  as  certain  of  these 
other  experiments  are  less  open  to  criticism  than  our  own.  The 
researches  of  Eisenberg  and  Volk,*  giving  definite  information  on 
the  relations  between  agglutinins  and  bacteria  and  leading  to  many 
new  and  important  experiments  on  this  subject,  are  particularly 
instructive  in  this  connection.  These  authors  found  that  the  law 
of  definite  proportions  does  not  apply  to  the  union  of  the  agglu- 
tinin  with 'the  agglutinable  substance  of  bacteria.  In  the  course 
of  their  work  they  noted  that  if  a  dose  A  of  bacteria  is  added  to 
a  given  dose  of  agglutinating  serum  the  results  are  the  same  as 
those  just  described  for  hemolytic  serum,  and  depend  on  whether 
A  is  added  all  at  once  or  in  divided  doses :  less  bacteria  are  agglu- 
tinated when  added  little  by  little.  Bacteria  can  absorb  much 
more  agglutinin  than  is  necessary  to  clump  them.  If  the  amount 
of  agglutinin  is  considerable,  an  equilibrium  is  established  between 
the  fraction  that  remains  free  and  the  one  absorbed  by  the  bac- 
teria, the  degree  of  saturation  depending  on  the  concentration  of 
the  agglutinin.  We  cannot  reproduce  here  in  detail  the  interest- 
ing conclusions  of  these  observers  (variations  in  the  coefficient  of 
absorption  in  proportion  to  the  dose  of  agglutinin,  the  function 
of  the  relative  concentrations  of  [agglutinin  and  agglutinable  sub- 
stances, etc.);  the  most  important  fact  is  that  the  agglutinable 
substance  absorbs  amounts  of  agglutinin  that  vary  according  to  the 
relative  proportions  of  the  two  reacting  substances. 

It  would  seem  legitimate,  then,  to  assume  that  the  law  of  fixed 
proportions  is  not  applicable  to  the  absorption  phenomena  by 
cells  or  bacteria  for  the  active  principles  of  specific  sera.  The  pro- 
portions would  seem  to  vary  as  markedly  as  do  the  conditions  of 
experiment.  The  conditions  are  very  unlike  those  met  with  in 
straight  chemistry,  which  depend  on  equations  and  equivalents. 
It  is  simply  for  the  purpose  of  expressing  this  idea  more  emphatically 
that  we  have  compared  these  phenomena  with  those  of  dyeing. 
*  Zeit.  fur  Hygiene,  Vol.  XL,  1902. 


ACTION  OF  ANTITOXINS  ON  TOXINS.  263 

Certain  observers  in  discussing  this  comparison  have  supposed 
that  we  overlooked  the  " chemical  nature"  of  the  combination 
of  active  serum  with  the  fixing  substance  of  the  corpuscle.  In 
other  words,  they  have  imagined  that  we  regard  this  fixation  as 
depending  entirely  on  mechanical  causes  (surface  adhesion,  etc.), 
to  the  exclusion  of  any  elective  or  specific  affinity.* 

To  read  these  authors  it  would  almost  appear  as  if,  in  our  opinion, 
corpuscles  absorb  the  active  substances  of  suitable  immune  sera 
indifferently  and  without  any  special  affinity,  as  charcoal  collects 
various  gases  indiscriminately!  We  have  never  committed  our- 
selves on  the  intimate  nature  of  the  reaction,  but  simply  as  to  its 
general  appearance.  The  expressions  *  purely  mechanical  causes" 
and  "  surf  ace  adhesion"  do  not  occur  in  our  descriptions.  We 
shall  not  go  any  further  into  the  discussions  as  to  whether  dyeing 
phenomena  should  be  qualified  as  " physical"  or  as  " chemical. " 
The  point  of  importance  is  that  these  reactions  differ  from  those 
of  ordinary  chemistry  in  that  they  are  not  expressed  by  equations; 
the  proportions  in  which  the  substances  unite  vary  according  to  the 
conditions  of  the  experiment.  It  is  simply  this  idea  of  a  variability 
in  proportions  which  led  to  our  comparison  and  which  to  our  think- 
ing would  justify  it.  The  results  of  Eisenberg  and  Volk,  which 
agree  with  our  own,  render  this  comparison  still  more  legitimate. 
That  in  the  case  of  specific  sera  and  susceptible  cells  we  have  to  do 
with  real  affinities  would  seem  evident  from  the  principle  of  specifi- 
city on  which  we  have  so  much  insisted  and  which  no  one  questions. 
It  is  certain  that  these  cells  show  a  truly  specific  and  exclusive 
avidity  for  their  appropriate  antibodies.  That  is  no  reason,  how- 
ever, for  their  absorption  in  fixed  proportions  nor  that  the  resulting 
compound  should  be  of  fixed  and  invariable  composition. 

Having  finished  this  digression  we  may  return  to  toxins  and 
antitoxins  and  reconsider  the  question  already  stated.  Does  the 
combination  of  these  elements  occur  in  fixed  and  constant  propor- 
tions, and  is  the  resulting  product  always  the  same,  or  may  the 
proportions  vary  within  wide  limits  and  the  resulting  compounds 

*  We  may  remark  in  passing  that  it  seems  unwise  to  assert,  as  some  authors  do, 
that  dyeing  phenomena  should  never  be  considered  from  a  chemical  standpoint, 
and  that  dyed  substances  absorb  dyes  in  a  purely  mechanical  manner,  owing  to 
physical  properties  (texture,  porousness),  and  never  owing  to  certain  chemical 
affinities  that  depend  on  their  composition. 


264  STUDIES  IN   IMMUNITY. 

differ  according  to  the  experimental  conditions,  and  contain  with  a 
given  dose  of  one  substance  variable  amounts  of  the  other? 

Before  seeking  an  answer  we  must  recall  certain  experimental 
data.  In  the  first  place  it  has  been  shown  that  if  an  amount  A  of 
antitoxin  is  necessary  completely  to  neutralize  an  amount  T  of  toxin, 
that  2A,  3A  or  nA  is  necessary  to  neutralize  2T,  3T  or  nT  respec- 
tively. If  we  regard  the  antitoxin  as  acting  directly  on  the  toxin 
and  combining  with  it,  there  is  nothing  surprising  in  this  fact.  It 
would,  moreover,  seem,  a  priori,  evident.  This  fact,  however, 
affords  no  definite  information  as  to  whether  antitoxin  and  toxin 
unite  in  definite  and  constant  proportions.* 

There  is  a  second  very  important  fact  mentioned  by  Ehrlich. 
Having  determined  very  exactly  the  minimal  lethal  dose  of  toxin, 
let  us  suppose  that  it  is  necessary  to  add  100  fatal  doses  of  this  toxin 
to  a  quantity  A  of  antitoxin  to  produce  a  neutral  mixture.  We 
admit  that  the  dose  A  of  antitoxin  is  just  sufficient  to  neutralize  the 
toxin  or,  in  other  words,  to  produce  a  harmless  mixture  containing 
no  excess  of  antitoxin.  Let  us  now  prepare  a  mixture  containing 
also  a  dose  A  of  antitoxin,  but  101  minimal  lethal  doses  of  toxin. 
We  might  suppose  that  this  mixture  would  kill  a  test  animal,  since 
it  contains  an  excess  of  toxin  equal  to  one  fatal  dose.  This  is  not 
what  happens,  however;  the  animal  shows  only  slight  symptoms. 

Mixtures  may  be  prepared  containing  a  considerably  increased 
amount  of  toxin,  even  as  much  as  200  lethal  doses,  to  the  same  dose 
A  of  antitoxin,  without  proving  fatal  for  animals  within  the  usual 
time  limit.  The  injection  of  such  mixtures  produces  slight  edemas 
if  the  excess  of  toxin  is  slight,  and  more  serious  ones  if  the  excess  is 
considerable. 

This  is  "  Ehrlich 's  phenomenon."  It  evidently  offers  a  notable 
objection  to  explanation  by  the  law  of  fixed  proportions.  The 
simplest  explanation  of  the  phenomenon  is  evidently  the  one  we 
have  already  mentioned,  namely,  that  the  antagonistic  substances 

*  It  would  seem  unnecessary  to  insist  on  this  fact,  if  it  were  not  that  a  bac- 
teriologist has  recently  asserted,  in  an  analogous  manner,  that  the  agglutinin  unites 
in  definite  proportions  with  the  agglutinable  substance  of  bacteria,  basing  the 
assertion  on  the  obvious  fact  that,  if  a  dose  A  of  serum  is  necessary  to  agglutinate 
a  dose  B  of  emulsion,  that  2A  is  necessary  to  produce  the  same  effect  on  2B.  If  we 
were  to  reason  in  this  way,  we  should  assert  that  a  paint  unites  in  definite  propor- 
tions with  the  surface  of  a  wall,  since,  if  a  quantity  A  of  paint  is  needed  to  paint 
10  square  meters,  2  A  is  necessary  to  paint  20  square  meters. 


ACTION   OF  ANTITOXINS  ON  TOXINS.  265 

(toxin  and  antitoxin)  combine  in  variable  proportions.  We  may 
imagine  that  each  molecule  of  toxin  is  able  to  unite  with  or  fix  a 
variable  number  of  molecules  of  the  antitoxin.  Let  us  suppose,  for 
example,  that  a  molecule  T  of  toxin  can  unite  either  with  a  single 
molecule  or  with  2,  3,  4  or  5  molecules  A  of  antitoxin.  Five 
compounds  then  are  possible  that  may  be  designated  TA1,  TA2, 
TA3,  TA4  and  TA5.  These  mixtures  will  be  more  or  less  toxic, 
according  to  the  amount  of  antitoxin  present.  The  first,  TA1  would 
be  rather  poisonous,  although  less  so  than  pure  toxin;  the  following 
TA2  and  TA3  would  be  successively  less  toxic;  TA4  and  TA5  may 
be  supposed  to  have  no  toxic  effect. 

If  we  were  to  mix  with  one  volume  of  toxin  containing  100  mole- 
cules (T)  a  volume  of  antitoxin  containing  200  molecules  (A),  we 
should  have  a  compound  TA2.  The  antitoxin  would  be  equally 
distributed  over  all  the  molecules  present  to  form  a  compound  of 
distinct  though  slight  toxicity.  This  compound  represents  the 
toxic  molecule  partially  saturated  with  antitoxic  molecules.  It  is 
an  attenuated  but  not  a  neutralized  toxin. 

If  to  the  same  number  of  toxin  molecules  (100  T)  we  were  to  add 
300  molecules  of  antitoxin,  the  compound  TA3  would  be  formed, 
and  so  on.  In  each  instance,  and  notwithstanding  the  dosage,  the 
antitoxin  will  be  equally  distributed  among  all  the  toxic  molecules. 
The  resultant  compounds  would  differ  according  to  the  amount 
of  antitoxin.  As  a  result,  we  would  never  find  in  such  mixtures 
quite  free  and  intact  toxin  in  conjunction  with  a  toxin  completely 
saturated  with  antitoxin. 

On  the  other  hand,  if  toxin  and  antitoxin  united  regularly  in  a 
fixed,  constant  and  uniform  proportion,  it  would  be  easy  to  obtain 
a  mixture  containing  both  intact  toxin  and  saturated  toxin  by 
adding  to  a  certain  volume  of  toxin  a  relatively  small  amount  of 
antitoxin. 

In  brief,  we  must  regard  a  mixture  of  toxin  with  an  incomplete 
dose  of  antitoxin*  as  one  or  the  other  of  two  very  different  com- 
pounds, in  accordance  with  whether  we  accept  the  hypothesis  of  a 

*  We  may  note  at  once  that  it  is  precisely  such  non-fatal  mixtures  that  give 
rise  to  Ehrlich's  phenomenon,  that  is,  mixtures  containing  a  dose  of  antitoxin 
capable  of  neutralizing  100  lethal  doses  of  toxin  plus  a  slight  excess  of  toxin,  say 
120  doses. 


266  STUDIES   IN   IMMUNITY. 

combination  in  fixed  proportions,  or  the  hypothesis  of  union  in 
variable  proportions.  In  the  first  instance  we  conceive  of  the 
fluid  as  containing  two  substances  —  free  active  toxin  and  saturated 
and  inactive  toxin.  On  the  second  supposition  we  conceive  of  the 
mixture  as  containing  a  single  substance  —  an  incompletely  satu- 
rated, non-neutralized,  or  simply  an  attenuated,  toxin. 

It  is  evident  that  non-identity  in  the  composition  of  the  mixtures 
would  lead  to  a  marked  difference  in  action  on  the  animal  body.  And, 
in  accordance  with  the  view  adopted  as  to  the  union  of  the  antago- 
nistic substance,  one  would  presuppose  very  different  harmful 
properties  in  a  given  fluid.  It  is  quite  understandable,  however, 
that  a  fluid  containing  attenuated  toxin  should  be  less  dangerous 
than  one  containing  a  certain  dose  of  intact  toxin  along  with  com- 
pletely neutralized  toxin.  Ehrlich's  phenomenon  would  appear 
easily  explicable  if  we  accept  the  idea  of  a  combination  in  variable 
proportions. 

The  preceding  statement  is  evidently  schematic,  and  it  is  the 
fault  of  such  schemes  to  be,  in  general,  too  dogmatic.  Nor  is  our  own 
working  hypothesis  free  from  this  criticism,  and  should  therefore  not 
be  taken  too  literally.  For  example,  it  would  imply  that  the  union 
of  toxin  and  antitoxin  is  a  simple  molecular  union;  it  also  of  neces- 
sity implies  that  our  two  substances  obey  the  law  of  multiple  pro- 
portions strictly  (as  that  the  dose  of  antitoxin  in  TA3  for  example, 
is  an  exact  multiple  of  that  in  TA).  As  a  result,  therefore,  in  order 
to  simplify  expression  we  may  look  on  each  substance  in  question 
as  one  elemental  particle,  as  one  molecule,  which  is  by  definition 
indivisible.  It  is  evident  that  our  purpose  is  not  to  determine 
whether  toxin  and  antitoxin  unite  by  a  union  of  molecules  or  by 
an  exchange  of  atoms,  nor  to  find  out  whether  we  are  dealing  with 
combinations  in  exactly  multiple,  or  simply  in  variable,  proportions. 
Such  problems  are  beyond  our  present  range,  and  experiment  is 
unable  to  solve  them. 

We  are  dealing  with  a  single  idea,  and  our  schema  has  been  used 
simply  to  express  this  idea.  If  the  hypothesis  of  a  union  in  variable 
proportions  is  exact,  the  essential  characters  of  the  reaction  would 
be  as  follows: 

1.  When,  to  a  given  amount  of  toxin,  antitoxin  is  added  in  an 
amount  that  does  not  suffice  completely  to  neutralize,  the  anti- 


ACTION  OF  ANTITOXINS  ON  TOXINS.  267 

toxin  molecules  are  not  monopolized  by  certain  of  the  toxin  mole- 
cules, whose  affinities  become  thereby  satisfied,  leaving  the  remain- 
ing toxic  units  intact.  On  the  contrary,  the  antitoxin  molecules 
are  shared  by  and  divided  equally  among  all  the  toxic  molecules 
present,  which  thenceforth  are  partially  saturated  and  lose  to  a 
certain  extent  their  original  toxicity. 

2.  The   phenomena  of  intoxication   caused   by  injecting  this 
compound  into  animals  may  not  be  the  same  as  those  produced 
by  a  mixture  of  neutralized  toxin  plus  intact  toxin. 

3.  Between  the  extremes  of  free  toxin  and  entirely  saturated 
or  innocuous  toxin,  all  transitions  or  stages  of  progressive  attenua- 
tion may  exist.     Each  time  that  a  mixture  of  toxin  and  antitoxin 
is  made  in  a  given  relation  the  same  degree  of  attenuation  will  be 
produced. 

Ehrlich,  as  we  know,  interprets  his  phenomenon  in  quite  a  dif- 
ferent way.  He  thinks  that  toxin  and  antitoxin  unite  in  fixed  pro- 
portions. To  harmonize  his  phenomenon  with  the  law  of  fixed 
proportions,  Ehrlich  makes  the  supposition  that  the  composition 
of  toxic  bouillon  is  very  complex;  that  it  contains,  indeed,  several 
poisons:  one,  an  active  poison,  is  the  toxin  properly  speaking; 
another,  less  dangerous  poison,  is  the  toxon. 

A  molecule  of  toxin  absorbs  as  much  antitoxin  as  does  a  mole- 
cule of  toxon.  In  this  respect  the  two  substances  are  equivalent. 
But  the  toxin  exceeds  the  toxon  in  the  energy  of  its  affinity  for, 
or  in  other  words  is  more  avid  of,  antitoxin.  In  order  to  neutralize 
completely  a  toxic  bouillon  enough  antitoxin  must  be  added  to 
neutralize  both  toxin  and  toxon.  But  if  an  additional  amount  of 
toxic  bouillon  is  added,  the  additional  toxin  that  it  contains  will 
seize  antitoxin  that  has  already  combined  with  the  original  toxon, 
and  so  liberate  this  latter  substance.  In  other  words,  if  to 
antitoxin  a  few  doses  of  toxin  in  excess  of  the  proper  amount 
for  neutrality  are  added  to  obtain  a  fluid  containing  no  free 
toxin,  there  is,  to  be  sure,  uncombined  toxon;  but  since  this  is 
relatively  harmless  the  animal  withstands  the  injection  of  such  a 
mixture. 

As  a  matter  of  fact  we  have  simplified  this  explanation  con- 
siderably, for  Ehrlich  has  attributed  an  extraordinarily  complicated 
composition  to  the  toxic  bouillon  in  order  to  make  his  theory  agree 


268  STUDIES  IN  IMMUNITY. 

fully  with  experiment.  The  explanation  is  unquestionably  ingen- 
ious but  the  existence  of  certain  of  the  substances,  particularly 
of  toxons,  is  purely  hypothetical.  The  question,  then,  is  still  an 
open  one. 

MODE  OF  ACTION  OF  ANTI-ALEXIN  ON  ALEXIN. 

We  might  have  expressed  the  preceding  elementary  ideas  some 
time  ago.  In  fact,  they  were  suggested  to  us  by  some  experiments 
we  did  on  the  neutralization  of  alexin  by  anti-alexin  in  1900  at  the 
Pasteur  Institute  (Professor  MetchnikofFs  service).  But  it  seemed 
well  to  render  the  study  more  complete  by  considering  the  effect 
of  certain  other  toxins  on  their  antitoxins.  We  have  not  as  yet 
been  able  to  carry  out  this  work.  We  shall  therefore  limit  our- 
selves here  to  a  consideration  of  the  facts  we  obtained  some  time 
ago  so  as  to  obviate  their  future  consideration. 

We  may  consider,  then,  the  effect  of  a  suitable  anti-alexin;  that  is 
to  say,  the  serum  (heated  to  55  to  56  degrees)  of  an  animal  of  species 
B  that  has  received  two  or  three  injections  of  fresh  serum  from 
species  A,  on  the  alexin  in  the  fresh  serum  of  A.* 

Having  mixed  these  two  antagonistic  sera  we  need  a  reagent 
capable  of  detecting  whether  or  not  free  alexin  is  present.  For  this 
purpose  we  use  red  blood  bells  well  sensitized  by  a  specific  hemolytic 
serum  (55  degrees).  We  know  that  alexin  is  absent  when  these 
cells  remain  intact. 

Such  an  experiment  naturally  comprises  a  control  mixture  con- 
taining the  same  factors  as  the  preceding  mixture,  but  with  heated 
serum  from  a  normal  animal  of  the  same  species  as  the  one  that 
furnished  the  anti-alexic  serum. 

As  may  be  imagined,  our  first  experiments  are  to  determine 
whether  the  reactions  of  our  antagonistic  sera  will  give  the  "Ehrlich 
phenomenon,"  which  is  so  difficult  to  reconcile  with  the  hypothesis 
of  neutralization  according  to  fixed  proportions.  In  the  following 
experiments  certain  precautions  must  be  taken  to  avoid  experi- 
mental error.  In  the  first  place  only  a  single  antitoxic  effect,  namely, 
the  one  against  the  alexin  under  consideration,  must  be  allowed  to 

*  An  anti-alexic  serum  must  be  heated  to  55  degrees  to  destroy  its  proper  alexin, 
in  a  study  of  this  sort.  The  only  alexin  in  the  experiment  should  be  in  the  form 
of  the  fresh  serum  of  the  animal  against  which  the  antiserum  is  active.  (See 
"  Homolytic  sera,  their  antitoxins,  etc,"  p.  186.) 


ACTION  OF  ANTITOXINS  ON  TOXINS.  269 

intervene.  As  sensitized  corpuscles  are  employed  the  anti-alexic 
serum  should  be  without  effect  on  the  sensitizer  used  for  these 
corpuscles.  The  heated  normal  serum  used  as  control  to  the  anti- 
alexin  or  the  sensitizer  should  have  no  particular  properties  and 
should  be  simply  an  inert  fluid.  And  of  course  the  absence  of  hemo- 
lytic  activity  in  the  heated  sera  should  be  controlled.  The  reagent 
employed  (sensitized  red  blood  corpuscles)  should  be  added  only 
in  very  small  doses  in  order  that  the  smallest  amounts  of  free  alexin 
may  be  detected. 

To  fulfill  these  conditions  we  use  the  following  materials:  As 
alexin,  fresh  guinea-pig  serum  is  employed.  As  anti-alexin,  the 
heated  serum  of  a  rabbit  that  has  been  given  injections  two  or 
three  times  of  fresh  normal  guinea-pig  serum.  For  sensitized  cor- 
puscles hen  red  cells  washed  in  salt  solution  and  treated  with  rabbit 
>  hen  serum,  56  degrees,  are  employed.  As  controls  of  the  anti- 
alexin  and  the  sensitizer,  normal  rabbit  serum,  55  to  56  degrees. 

In  short,  the  toxin  (alexin)  is  from  the  guinea-pig.  The  other 
sera  (antitoxin,  sensitizer  and  control)  are  all  from  the  rabbit  and 
have  no  effect  on  one  another.  No  accessory  reactions,  therefore, 
should  destroy  the  accuracy  of  the  experiment. 

The  strength  of  the  anti-alexin  is  first  defined  by  the  following 
experiment : 

Tube  a.  Alexin,  0.2  of  a  cubic  centimeter;  anti-alexin  (55  degrees),  0.3  of  a 
cubic  centimeter. 

Tube  b.  Alexin,  0.2  of  a  cubic  centimeter;  normal  rabbit  serum  (55  degrees), 
0.3  of  a  cubic  centimeter. 

An  hour  or  two  later  0.2  of  a  cubic  centimeter  of  sensitizer  (serum 
rabbit  >  hen,  56  degrees)  is  added  to  each  tube  and  then  one  drop 
of  washed  hen  blood.  The  corpuscles  are  hemolyzed  in  20  minutes 
in  "6. "  There  is  no  hemolysis  in  "a,"  even  on  the  following  day. 

This  lack  of  hemolysis  in  "a"  may  be  shown  to  be  due  solely  to 
a  neutralization  of  the  alexin.  The  sensitizer,  present  in  relatively 
large  amount  in  respect  to  the  corpuscles,  is  unaffected.* 

Tube  a.     Sensitizer,  0.1  c.c;  anti-alexin,  2  c.c ;  fresh  rabbit  serum  (alexin),  2  c.c. 
Tube  6.     Same  as  "a,"  with  2  c.c.  normal  rabbit  serum,  55  degrees,  replacing 
the  anti-alexin. 

*  This  is  shown  by  replacing  the  guinea-pig  alexin,  which  is  specifically  affected 
by  the  anti-alexin,  by  another  alexin,  say  from  the  rabbit,  on  which  the  anti-alexin 
has  no  effect. 


270  STUDIES  IN  IMMUNITY. 

Tube  c.  Rabbit  alexin  2  c.c;  normal  rabbit  serum,  55  degrees,  2  c.c.  (Same 
as  "b"  without  sensitizer.) 

To  each  tube  is  added  one  drop  of  hen  blood.  Hemolysis  occurs  rapidly  in  "  a  " 
and  "b."  It  is  only  partial  in  "c"  after  3  hours.  It  is  evident,  then,  that  the 
anti-alexin  can  have  no  effect  on  the  sensitizer,  which,  in  the  experiment,  is  only 
one-twentieth  of  the  volume  of  the  antiserum.  It  is  evident,  too,  that  the  sensitizer 
acts  energetically  although  present  in  small  amount  and  diluted  in  4  c.c.  of  fluid. 
In  the  rest  of  the  article,  for  the  sake  of  simplicity,  these  control  experiments  need 
not  be  insisted  on  in  each  instance. 

We  then  determine  the  potency  of  the  anti-alexin.  For  this 
purpose  varying  amounts  of  alexin  (say  from  0.05  to  1.2  c.c.)  are 
added  to  a  constant  volume  (0.3  of  a  cubic  centimeter)  of  anti- 
alexic  or  of  normal  serum  (55  degrees).  The  following  tubes  are 
prepared : 

A.  Tubes  containing  0.3  of  a  cubic  centimeter  of  anti-alexin: 
a.  Alexin,   0.05  c.c.;  6.  Alexin,  0.1  c.c.;  c.  0.2  c.c.;  d.  0.3  c.c.; 

e.  0.4  c.c.;  /.  0.5  c.c.;  g.  0.6  c.c.:  h.  0.7  c.c.;  i.  0.8  c.c.;  j.  0.9  c.c.; 
k.    1.2  c.c. 

B.  Same  as  preceding  tubes,  with  heated  normal  rabbit  serum 
(non-anti-alexic)  replacing  anti-alexin. 

To  each  tube  is  then  added  0.2  of  a  cubic  centimeter  of  sensitizer 
and  an  hour  later  one  drop  of  a  suspension  of  hen  corpuscles  in 
NaCl.  The  mixtures  are  kept  at  room  temperature  (18°  C.). 

In  the  tubes  that  do  not  contain  anti-alexin,  hemolysis  takes 
place  rapidly  in,  say,  15  minutes  in  those  tubes  containing  most 
alexin,  and  in  30  minutes  in  the  one  with  the  smallest  amount. 
Consequently  0.05  of  a  cubic  centimeter  of  alexin  represents  the 
minimal  dose  for  hemolysis  in  a  half  hour,  which  may  be  taken  as 
a  unit.  We  may  add  that  the  dilution  of  this  small  amount  of 
alexin  in  heated  serum,  one  volume  of  alexin  in  ten  of  heated  serum, 
for  the  sake  of  mensuration  has  no  effect  on  its  activity. 

We  may  now  consider  the  tubes  containing  anti-alexin.  Let 
us  consider  first  the  final  reading,  say  on  the  following  day,  in  order 
to  allow  for  the  maximal  action  of  the  smallest  amounts 'of  free 
alexin.  On  the  following  day  hemolysis  is  complete  in  the  tubes 
containing  0.5  of  a  cubic  centimeter  or  more  of  alexin;  it  is  partial 
in  the  0.4  c.c.  tube  and  slight  in  the  0.3  c.c.  tube.  Two-tenths  of 
a  cubic  centimeter  or  less  shows  no  hemolysis. 

It  may  be  noted  that  such  results  argue  somewhat  against  the 
hypothesis  of  a  combination  in  fixed  proportion.  If  there  is  slight 


ACTION  OF  ANTITOXINS   ON  TOXINS.  271 

hemolysis  in  tube  "d,"  it  is  due  to  an  almost  negligible  trace 
of  free  alexin;  the  next  mixture,  "e,"  which  contains  two  addi- 
tional minimal  doses  of  alexin,  should  show  complete  hemolysis  but 
actually  does  show  only  partial  hemolysis.  Ehrlich's  phenomenon, 
then,  is  present,  but  is  not  well  marked.  It  is  also  to  be  noted  that 
a  volume  of  anti-alexin  equal  to  that  of  the  alexin  must  be  used  in 
order  to  protect  corpuscles  effectively. 

The  most  interesting  results  are  evident  soon  after  the  mixtures 
are  made,  and,  if  the  moment  when  hemolysis  is  complete 
is  noted,  Ehrlich's  phenomenon  is  then  very  striking.  The  mix- 
ture of  1.2  c.c.  of  alexin  and  0.3  of  a  cubic  centimeter  of  anti- 
alexin  should,  according  to  the  hypothesis  of  neutralization  in 
fixed  proportions,  contain  0.3  of  a  cubic  centimeter  of  neutralized 
alexin  and  0.9  of  a  cubic  centimeter  of  intact  alexin.  In  such  a 
mixture  hemolysis  is  complete  only  after  70  minutes,  which  is  at 
least  twice  as  long  as  is  required  in  a  mixture  containing  a  single 
fatal  dose  of  alexin  without  antitoxin.  Hemolysis  is  complete  in 
this  latter  mixture  before  it  begins  in  the  former. 

In  mixtures  of  anti-alexin  with  doses  of  alexin  from  0.4  to  0.9 
of  a  cubic  centimeter,  corpuscles  are  eventually  hemolyzed,  the 
rapidity  in  beginning  and  completion  of  the  process  varying  directly 
with  the  amount  employed.  For  example,  although  hemolysis  is 
complete  in  70  minutes  in  a  tube  with  1.2  c.c.  of  alexin,  there  is 
none  at  this  time  with  a  dose  of  0.9  of  a  cubic  centimeter 
("/,"),  2|-  hours  being  necessary  for  complete  hemolysis,  and  at 
2J  hours  hemolysis  is  only  partial  in  tube  "i,"  has  barely  begun  in 
tube  "  h, "  and  so  on.  In  short,  the  time  necessary  for  the  liberation 
of  hemoglobin  varies  indirectly  with  the  amount  of  alexin  employed. 

The  experiment  shows  that  a  dose  of  anti-alexin  that  can  com- 
pletely neutralize  6  fatal  doses  of  alexin  (one-half  hour)  mil  check 
24  fatal  doses  to  such  an  extent  that  they  produce  less  rapid  hemoly- 
sis than  a  single  unaffected  fatal  dose.  It  is  impossible,  therefore, 
to  imagine  that  anti-alexin  added  to  a  large  close  of  alexin  completely 
neutralizes  part  of  it,  leaving  the  excess  free:  such  mixtures  do  not 
act  at  all  as  do  simple  dilutions  of  alexin. 

Anti-alexin  affects  all  the  alexin  present  equally,  in  accordance  with 
a  law  of  varying  proportions,  as  has  just  been  fully  explained. 
Ehrlich's  explanation  for  diphtheria  toxin,  presupposing  the  exist- 


272  STUDIES   IN   IMMUNITY 

ence  of  toxons,  will  not  explain  the  results  just  considered.  This 
is  evident  in  considering  the  proportions  of  sera  employed,  the 
fact  that  the  minimal  dose  of  alexin  (0.05  c.c.)  is  strongly  hemolytic, 
and  the  fact  that  the  time  necessary  for  the  appearance  of  hemoly- 
sis  decreases  regularly  and  gradually  with  the  amount  of  alexin 
employed. 

The  conclusion,  then,  is  that  each  of  the  alexin-antialexin  mix- 
tures forms  a  new  substance  or  complex  containing  neither  of  the 
antagonistic  substances  in  pure  state,  but  depending  for  composition 
on  the  relative  proportion  of  each  substance  employed.  The  com- 
plex is  different  in  each  successive  mixture,  being  more  or  less  toxic 
in  accordance  with  the  degree  of  saturation  of  the  toxin  by  the 
antitoxin.  The  anti-alexin  attenuates  the  alexin  until  it  com- 
pletely neutralizes  it  if  the  dose  is  sufficient. 

It  follows  from  this  experiment  that  it  is  impossible  to  prepare 
an  exactly  neutral  mixture  of  alexin  and  anti-alexin,  that  is  to  say, 
a  mixture  that  is  absolutely  non-toxic  and  non-antitoxic.  This 
conception,  to  be  sure,  is  not  novel,  but  is  simply  a  restatement  of 
Ehrlich's  phenomenon.  Take,  for  example,  a  mixture  containing  a 
moderate  dose  of  alexin  ("/"  or  "g")  in  addition  to  the  anti-alexin. 
Such  a  mixture  is  toxic,  since  the  corpuscles  finally  hemolyze. 
It  is  also  antitoxic,  since  similar  mixtures  containing  the  same 
amount  of  anti-alexin,  but  more  alexin,  are  simply  hemolyzed  more 
slowly.  Such  a  result  is  a  corollary  to  the  idea  of  a  combination 
in  variable  proportions,  according  to  which  a  whole  series  of  degrees 
of  progressive  attenuation  between  an  active  toxin  and  a  neutral- 
ized toxin  may  be  formed. 

The  proof  that  such  a  mixture  contains  a  distinct  anti-alexic 
power  in  addition  to  a  real  toxicity  lies  in  comparing  it  with  a  similar 
mixture  containing  a  little  more  alexin,  in  which  case  hemolysis 
is  very  slow.  A  more  direct  proof  might  be  preferable.  We  may 
prepare  such  a  mixture,  supposed  to  be  at  once  toxic  and  anti- 
toxic, by  mixing  0.3  of  a  cubic  centimeter  of  anti-alexin  with  0.5 
of  a  cubic  centimeter  of  alexin.*  As  a  control  a  mixture  of  the 
same  amounts  of  non-antialexic  serum  (normal  rabbit  serum,  56 
degrees)  and  inactive  alexic  serum  (56  degrees)  is  prepared.  Two 
or  three  hours  later  a  little  active  alexin  (0.1  c.c.)  is  added  to  each 
*  In  such  an  instance  hemolysis  takes  place  after  several  hours. 


ACTION   OF  ANTITOXINS  ON  TOXINS.  273 

tube.  It  may  be  presupposed  that  this  substance  will  be  distinctly 
attenuated  in  the  first  mixture,  and  such  proves  to  be  the  case 
although  the  result  is  not  very  striking.  If  sensitized  hen  cor- 
puscles are  subsequently  added  to  these  tubes,  hemolysis  is  more 
rapid  in  the  control  than  in  the  first  tube,  although  the  difference 
is  not  extremely  marked.  And  why? 

The  first  mixture  contains  in  addition  to  the  anti-alexin  a  total 
of  0.6  of  a  cubic  centimeter  of  alexin  added  in  two  successive  frac- 
tions. A  comparison  of  the  hemolytic  power  of  this  mixture  with 
that  of  a  mixture  containing  0.6  of  a  cubic  centimeter  of  alexin 
added  in  a  single  dose  is  interesting. 

We  prepare,  then,  liquid  A,  containing  0.3  of  a  cubic  centimeter 
of  anti-alexin  plus  0.5  of  a  cubic  centimeter  of  alexin.  Three  hours 
later  we  add  0.1  of  a  cubic  centimeter  of  alexin  to  it  and  at  the 
same  time  prepare  mixture  B,  which  contains  0.3  of  a  cubic  cen- 
timeter of  anti-alexin  plus  0.6  of  a  cubic  centimeter  of  alexin.  We 
then  add  to  each  mixture  0.2  of  a  cubic  centimeter  of  rabbit  > 
hen  sensitizer  and  an  hour  later  two  drops  of  hen  blood  to  each 
tube.  Hemolysis  requires  an  hour  in  tube  A,  and  an  hour  and  three 
quarters  in  B.  An  anti-alexic  serum  then  neutralizes  a  given 
dose  of  alexin  better  when  it  is  added  all  at  once  than  when  it  is 
added  in  successive  fractions.  This  experiment  recalls  the  one  in 
which  red  blood  corpuscles  are  added  to  a  hemolytic  serum  either 
all  at  once  or  in  divided  doses. 

In  a  mixture  of  alexin  with  anti-alexin  the  latter  substance 
is  uniformly  distributed  over  all  the  alexin,  and  all  the  toxic  mole- 
cules are  equally  modified ;  the  composition  of  the  mixtures  is  homo- 
geneous. But  if  we  subsequently  add  more  alexin  to  such  a  mixture 
it  tends  to  remove  the  antitoxin  from  the  combination  into  which 
it  has  entered,  tends,  in  other  words,  to  break  up  its  established 
distribution.  For  example,  if  the  original  complex  is  TA2,  the 
addition  of  another  T  would  tend  to  form  2TA.  But  since  the 
combination  TA2  is  already  formed,  a  new  reaction  (i.e.,  removal 
of  the  anti-alexin  from  the  complex)  is  somewhat  difficult  to  bring 
about,  and  the  additional  dose  of  alexin,  in  consequence,  is  not  read- 
ily attenuated.*  In  other  words,  TA2  does  not  give  a  part  of  its 

*  If  all  the  alexin  had  been  added  at  once,  the  complex  TA  would  of  course 
have  been  formed. 


274  STUDIES  IN   IMMUNITY 

antitoxin  unresistingly.  The  difficulty  in  such  a  reaction  naturally 
depends  on  the  toxins  and  antitoxins  under  consideration,*  and  it 
is  probable  that  the  stability  of  the  complex  will  vary  in  different 
instances.  If  the  complex  is  very  stable,  an  additional  T  will  remain 
intact;  on  the  other  hand,  it  is  readily  attenuated  when  the  complex 
is  unstable. 

It  would  seem  quite  evident  from  a  recent  experiment  of  Mor- 
genroth'sf  that  these  various  possibilities  may  well  exist. 

This  investigator  sensitized  well-washed  red  blood  cells  with 
their  specific  serum  (55  degrees),  centrifugalized,  removed  the 
supernatant  fluid  and  washed  the  corpuscles  several  times  in  salt 
solution.  The  resulting  sensitized  corpuscles  were  suspended  in 
a  medium  that  contained  no  free  sensitizer.  To  such  a  suspension 
he  then  added  normal  unsensitized  corpuscles  of  the  same  sort. 
If  alexin  in  moderate  amount  is  immediately  added  to  such  a  mix- 
ture, only  part  of  the  corpuscles  are  destroyed,  namely,  only  those 
that  were  sensitized.  But  if  some  time  elapses  before  the  alexin 
is  added,  all  the  corpuscles  are  hemolyzed  indiscriminately.  Mor- 
genroth  very  properly  draws  the  conclusion  that  the  normal  cor- 
puscles are  able  after  a  certain  interval  to  remove  a  certain  amount 
of  sensitizer  from  the  sensitized  corpuscles.^  In  other  words,  a 
change  in  distribution  of  the  sensitizer  analogous  to  the  change  in 
distribution  of  the  anti-alexin  in  the  preceding  experiment  has 
taken  place. 

It  is  evident  that  the  complex  formed  by  the  union  of  the  sen- 
sitizer with  the  fixing  substance  of  the  cell  (which  may  be  designated 
CS2)  gives  up  a  part  of  its  sensitizer  to  other  cells  rather  easily  and 
becomes,  say,  CS.  In  this  instance  the  complex  is  rather  unstable. 
We  may  compare  this  result  with  the  one  noted  at  the  beginning 
of  this  article,  which  dealt  with  the  effect  of  adding  alexin  in  a  single 
or  in  divided  doses  to  sensitized  cells,  in  which  instance  the  com- 
plex "  cell-sensitizer-alexin "  (or,  better,  stroma-alexin)  is  remark- 
ably stable.  Destroyed  blood  cells  laden  with  alexin  do  not  yield 

*  And  even  with  a  given  toxin  and  antitoxin  the  more  or  less  complete  satura- 
tion of  the  complex  must  be  considered.  A  complex  TA*  would  probably  give 
up  its  antitoxin  more  readily  than  does  TA*. 

f  Munch,  med.  Wochenschrift,  1903,  No.  2. 

I  It  is  also  quite  probable,  as  Morgenroth  states,  that  the  fluid  serves  as  a 
medium  of  passage  for  the  sensitizer  from  one  cell  to  the  other. 


ACTION  OF  ANTITOXINS  ON  TOXINS.  275 

it  to  other  corpuscles  even  when  they  are  strongly  sensitized.  This 
justifies  the  preceding  remarks  as  to  the  varying  stability  of  com- 
plexes obtained.* 

It  is  evident  that  for  these  reasons  and  from  these  experiments 
we  believe  in  the  hypothesis  of  a  combination  in  variable  propor- 
tions. It  is  well,  however,  to  verify  this  hypothesis  in  its  more 
important  bearings. 

Two  different  standpoints  may  be  assumed  in  determining  the 
toxicity  of  any  poisonous  substance.  In  the  first  place  an  estima- 
tion of  the  strength  of  a  poison  may  be  made  by  determining  how 
many  cells  or  what  weight  of  animal  it  will  destroy.  On  the  other 
hand,  the  toxicity  in  relation  to  the  time  necessary  to  accomplish 
a  given  result  may  be  determined. 

In  considering  a  mixture  of  alexin  and  anti-alexin,  let  us  mix 
0.3  of  a  cubic  centimeter  of  anti-alexin  with  1.2  c.c.  of  alexin 
(guinea-pig).  This  1.5  c.c.  of  fluid  (A)  contains,  according  to  our 
hypothesis,  only  attenuated  alexin,  and  only  slightly  attenuated  at 
that,  as  the  dose  of  anti-alexin  is  small.  In  other  words,  the  anti- 
alexin  has  not  neutralized  part  of  the  alexin  and  left  the  rest  un- 
altered. We  have  then  to  deal,  not  with  a  simple  quantitative 
diminution  of  the  alexin,  but  with  a  complex  that,  as  a  whole,  is 
less  toxic,  in  that  it  hemolyzes  even  a  small  dose  of  corpuscles 
remarkably  slowly. 

Since  all  the  alexin  has  been  transformed,  but  none  of  it,  properly 
speaking,  destroyed,  and  since  we  are  dealing,  not  with  a  quantita- 
tive diminution,  but  with  a  modification  of  the  totality  of  alexin, 
it  is  conceivable  how  the  mixture  can  destroy  a  relatively  consider- 
able amount  of  red  blood  cells,  although  the  hemolysis  may  be  slow. 

*  An  idea  of  Morgenroth's  phenomenon  may  be  gained  from  a  certain  staining 
phenomenon  that  would  appear  in  a  rough  way  to  be  suggestively  analogous. 
We  place  about  half  of  a  strip  of  filter  paper  at  the  bottom  of  a  crystallizing  dish 
and  pour  over  it  a  little  solution  of  methylene  blue.  In  a  short  time  the  paper 
removes  all  the  color  from  the  fluid.  We  then  take  two  more  bits  of  filter  paper  and 
place  one  of  them  (A)  at  the  bottom  of  the  dish  and  the  other  (B)  very  near  the 
original  strip  that  has  been  lying  in  the  solution  and  that  has  absorbed  the  blue. 
Care  is  taken  that  B  does  not  actually  touch  the  original  paper.  B  soon  becomes 
more  colored  than  A.  The  first  strip  is  decolorized  at  the  point  nearest  to  B. 
The  distribution  of  color  between  this  original  strip  and  B  tends  to  homogeneity, 
as  does  the  sensitizer  in  Morgenroth's  experiment  tend  to  be  equally  shared  by 
all  the  cells  present. 


276  STUDIES  IN   IMMUNITY. 

We  may  now  compare  mixture  A,  that  contains  much  attenuated 
alexin,  with  a  second  mixture  B  having  the  same  volume,  but  a 
different  constitution.  This  second  mixture  has  been  obtained  by 
adding  a  very  small  amount  of  normal  alexin  to  an  inactive,  cer- 
tainly not  anti-alexic  serum  (normal  guinea-pig  or  rabbit  serum, 
55  degrees).  The  mixture,  then,  is  a  simple  dilution  of  active  alexin 
in  an  inert  fluid.  For  example,  B  may  be  a  mixture  of  0.3  of  a  cubic 
centimeter  of  heated  normal  rabbit  serum  plus  0.1  of  a  cubic  cen- 
timeter of  alexin  diluted  in  1.1  c.c.  of  the  same  serum  heated  to 
55  degrees. 

It  is  evident  that  small  amounts  of  sensitized  hen  corpuscles 
added  to  mixture  B  will  be  rapidly  destroyed  owing  to  the  presence 
of  the  active  alexin.  But  since  the  amount  of  this  alexin  is 
relatively  inconsiderable,  it  is  evident  that  if  much  sensitized  blood 
is  added  hemolysis  even  on  long  standing  will  be  only  partial. 

As  will  be  suspected,  the  two  mixtures  A  and  B,  containing  each 
the  same  volume  and  both  capable  of  producing  hemolysis,  are  in 
reality  endowed  with  very  different  properties.  If  the  hemolytic 
property  of  the  mixtures  is  estimated  by  means  of  a  small  dose  of 
sensitized  corpuscles,  e.g.,  one  drop,  B  will  appear  more  active  than 
A;  that  is  to  say,  it  will  hemolyze  a  few  corpuscles  more  rapidly. 
But  the  opposite  result  will  be  obtained  if  the  hemolytic  power  is 
measured  by  the  total  amount  of  corpuscles  that  each  will  destroy. 
For  example,  if  a  large  dose  of  sensitized  blood  is  added  (say, 
1.5  c.c.)  on  the  following  day  it  will  be  found  that  most  of  the 
corpuscles  in  B  are  intact,  whereas  all  the  corpuscles  in  A  are 
hemolyzed.  In  other  words,  the  alexin  is  quantitatively  greater 
in  A,  and  qualitatively  more  active  (rapidity  of  action)  in  B. 

These  experimental  results  agree  with  our  hypothesis.  It  is  no 
longer  possible  to  assume  that  an  insufficiently  neutralizing  dose  of 
anti-alexin  changes  alexic  serum  to  a  mixture  of  perfectly  neutral- 
ized and  of  intact  alexin.  If  it  were,  the  resultant  fluid  would  be 
simply  a  dilution  of  normal  alexin  in  a  certain  amount  of  inert 
fluid,  in  other  words,  a  mixture  identical  with  B. 

It  would  not  be  legitimate  to  draw  too  generalized  conclusions 
from  these  experiments  on  alexin  and  anti-alexin ;  the  further  study 
of  various  toxins  and  antitoxins,  from  the  point  of  view  of  method 


ACTION  OF  ANTITOXINS  ON  TOXINS.  277 

of  combination,  is  necessary.  We  may,  however,  note  that  certain 
well-known  hitherto  enigmatic  facts  become  easily  explicable  on 
the  hypothesis  of  union  in  variable  proportions.  One  or  two  exam- 
ples may  be  given : 

It  has  been  noted  (particularly  with  tetanus)  that  a  toxin-anti- 
toxin mixture  that  is  harmless  for  an  animal  of  species  A  shows 
evident  toxicity  for  an  animal  of  species  B.  Instances  of  this 
sort  have  been  mentioned,  particularly  by  Buchner  and  by  Roux 
and  Vaillard.  Such  apparently  peculiar  results  are  an  almost 
necessary  corollary  to  our  hypothesis.  A  mixture  of  toxin  with 
even  a  weak  dose  of  antitoxin  contains  no  primitive  toxin,  but  in 
its  place  an  attenuated  toxin,  a  new  complex  endowed  with  in- 
dividual characteristics,  and  it  will  not  necessarily  act  in  the  same 
manner  on  different  animals.  It  is  reasonable  to  anticipate  that 
its  toxic  power  will  be  so  attenuated  as  to  produce  no  trouble  with 
certain  animals,  but  distinct  effect  on  others;  why,  indeed,  should 
they  all  react  alike  to  this  new  compound?  It  is  even  theoretically 
conceivable  that  completely  saturated  toxin  should  poison  certain 
species  or  certain  individuals.  According  to  our  conception  there 
is  no  sharp,  radical  difference  between  attenuation  and  neutraliza- 
tion of  a  toxin,  or,  in  other  words,  there  is  no  absolute  neutraliza- 
tion. There  is  simply  a  greater  or  less  degree  of  attenuation  in 
accordance  with  the  more  or  less  complete  saturation  of  the  toxin 
by  antitoxin.  This  saturation  is  often  practically  equivalent  to 
a  neutralization.  The  expression  "neutralization"  implies  that 
the  toxin  has  become  irrevocably  inactive  for  very  sensitive  animals 
owing  to  a  radical  abolition  of  the  substance  that  renders  it  toxic. 
With  such  complete  neutralization  the  degree  of  a  given  animal's 
susceptibility  is  not  a  significant  factor  in  the  result.  When, 
however,  an  apparent  neutralization  is,  in  reality,  only  a  very  marked 
attenuation,  the  degree  of  susceptibility  must  be  taken  into  account, 
as  the  attenuation  may  be  very  marked  as  far  as  animal  A  is  con- 
cerned, but  very  little  for  animal  B.  The  conception  of  variation 
in  attenuation,  then,  must  constantly  take  into  account  the  relation 
between  the  sensitivity  of  the  animal  body  and  the  harmful  nature 
of  the  substance  under  consideration. 

It  is  quite  comprehensible,  then,  how  a  mixture  that  is  rich  in 
antitoxin  (i.e.,  with  the  toxin  well  saturated)  and  inoffensive  for 


278  STUDIES  IN  IMMUNITY. 

healthy  animals  may  yet  be  dangerous  for  animals  that  are  weak- 
ened and  so  rendered  more  sensitive.  It  may  be  possible  to  ex- 
plain in  this  manner  the  intoxication  by  means  of  diphtheria  toxin 
of  animals  that  are  actually  producing  active  diphtheria  anti- 
toxin. Although  the  modification  of  toxin  is  still  going  on,  the 
animal  may  have  become  very  sensitive  to  even  a  saturated  toxin, 
which,  of  course,  is  without  effect  on  normal  animals.  The  sus- 
ceptibility of  guinea-pigs  immunized  against  the  cholera  vibrio  to  a 
tetanus  toxin-antitoxin  mixture  (Roux  and  Vaillard)  is  apparently 
another  example  of  this  fact.* 

A  relatively  small  amount  of  anti-alexin  suffices  to  protect  normal 
unsensitized  corpuscles  from  alexin,  but  a  large  amount  is  neces- 
sary to  protect  sensitized  blood  cells.  As  Morgenroth  and  Sachsf 
have  already  shown,  more  anti-alexin  is  necessary  to  protect  heavily 
sensitized  corpuscles  than  slightly  sensitized  ones;  in  other  words, 
the  toxicity  of  the  alexin-antialexin  complex  depends  on  the 
strength  of  the  sensitization. 

This,  leads  us  to  a  consideration  of  results  obtained  by  investi- 
gators on  the  injection  of  a  toxin  that  is  partially  neutralized  by 
antitoxin.  As  we  know,  such  a  substance  is  simply  attenuated 
toxin,  inoffensive  for  certain  animals,  but  distinctly  harmful  for 
others.  Dryer  and  Madsen  have  shown  that  such  an  incom- 
pletely saturated  mixture  of  diphtheria  toxin,  which  is  quite  in- 
nocuous for  the  guinea-pig,  produces  slight  symptoms,  (edema,  etc.) 
in  the  rabbit.  The  toxicity  of  such  a  mixture  might  be  increased 
by  varying  the  respective  amounts  of  the  antagonistic  substances 
and  so  preparing  a  slightly  less  attenuated  mixture.  Under  such 
conditions  an  animal  that  had  only  shown  edema  would  show 
distinct  poisoning.  Such  results,  indeed,  were  obtained  by  Dryer 
and  Madsen. 

These  investigators  consider  that  such  incompletely  saturated 
mixtures  contain  only  toxon,  that  is  to  say,  a  different  and  less  active 
substance  than  the  true  toxin.  This  toxon  seems  to  us  to  be  simply 
our  complex  —  toxin  incompletely  saturated  with  antitoxin.  Such 
a  complex  has  all  the  characteristics  attributed  to  toxons:  it  is  by 
definition  less  toxic  than  free  toxin  and  also  less  avid  of  antitoxin, 

*  Such  mixtures,  of  course,  are  inactive  for  normal  guinea-pigs. 

f  See  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  and  Sons,  p.  250. 


ACTION   OF   ANTITOXINS   ON  TOXINS.  279 

since  its  affinity  for  it  is  already  partially  satisfied.  It  is  evidently 
related  to  toxin  as  far  as  origin  and  composition  are  concerned, 
but  still  is  a  new  substance  and  therefore  gives  rise  to  different 
symptoms  than  a  small  amount  of  diluted  toxin.  Since  it  contains 
the  toxic  radical  its  injection,  of  course,  leads  to  formation  of  anti- 
toxin. For  Dryer  and  Madsen  have  shown  that  antitoxin  can  be 
produced  by  injections  of  the  substance  they  call  toxon.* 

We  may  add  further,  since  we  consider  the  existence  of  toxons 
as  distinct  substances  as  improbable,  that  we  do  not  wish  in  any 
way  to  cast  doubt  on  Ehrlich's  results  as  to  the  spontaneous  weak- 
ening of  toxins  by  conservation  and  the  corresponding  formation  of 
toxoids  from  them.  We  regard  the  effect  of  antitoxins  simply  as 
one  factor  in  the  attenuating  of  toxins,  but  there  may  well  be  others, 
such  as  oxygen  and  light,  the  exact  effect  of  which  is  not  well 
determined,  although  their  weakening  action  on  toxin  is  well 
defined. 

*  Zeitschrift  f.  Hygiene.,  XXXVII,  1901. 


XIV.    THE   PROPERTIES  OF  ANTISENSITIZERS   AND 
THE   CHEMICAL  THEORIES   OF   IMMUNITY.* 

BY  DR.   JULES   BORDET. 

One  of  the  most  important  problems  in  the  study  of  immunity, 
and  it  must  be  confessed  one  of  the  most  difficult  of  solution,  is 
the  specificity  of  serum.  The  problem  is,  of  necessity,  complex. 
We  have  known  for  some  time  that  the  antibodies  of  immune  sera 
are  specific  in  the  sense  that  they  affect  certain  substances  and 
do  not  affect  others.  But  in  addition  to  this  specificity  of  action 
we  apparently  must  recognize  a  specificity  of  origin.  For  example, 
a  given  sensitizer  (amboceptor  or  fixateur)  should  be  designated, 
not  only  in  respect  to  the  blood  corpuscles  or  bacterium  that  it 
attacks,  but  also  in  respect  to  the  animal  species  that  has  formed  it. 
Two  sensitizers,  both  active  against  the  cholera  vibrio,  but  derived 
in  one  case  from  the  rabbit  and  in  the  other  from  the  guinea-pig,  do 
not  act  exactly  alike  under  all  circumstances;  the  same  statement 
holds  for  antitoxic  sera.  We  know,  for  example,  that  the  duration 
of  a  passive  immunity  given  by  a  preventive  serum  varies,  depend- 
ing on  whether  the  serum  is  derived  from  the  same  species  as  the 
recipient  or  from  an  alien  species. 

A  study  of  antisensitizers  is  particularly  useful  as  throwing 
light  on  the  specificity  of  sera,  and  particularly  as  to  whether  this 
specificity  is  as  absolute  as  would  appear.  It  would  seem,  a  priori, 
reasonable  to  suppose  that  the  law  of  specificity  should  be  most 
evident  in  dealing  with  these  antisensitizers  or,  more  generally 
speaking,  in  dealing  with  anti-antibodies.  In  this  instance  we 
deal  with  substances  that  are  not  only  antibodies,  but  are  specific 
both  as  regards  action  and  origin.  It  would  seem  reasonable,  then, 
that  they  should  afford  the  most  notable  and  instructive  instances 
of  specificity. 

*  Les  proprie'te's  des  antisensibilisatrices  et  les  theories  chimiques  de  1'immu- 
nite".  Annales  de  1'Institut  Pasteur,  XVIII,  1904,  593. 

280 


PROPERTIES  OF  ANTISENSITIZERS.  281 

Antisensitizers  may  be  demonstrated  in  the  blood  of  the  animals 
that  have  been  vaccinated  against  a  hemolytic  serum.  Following 
the  work  of  Camus  and  Gley  and  Kossel  on  the  antitoxin  to  eel 
serum,  we  demonstrated  in  1899  *  that  the  injection  of  an  animal  of 
species  A  (rabbit)  with  the  serum  of  species  B  (hen)  gave  rise  to 
a  property  in  the  serum  of  A  that  neutralizes  the  hemolytic  effect 
of  serum  B  on  the  corpuscles  of  A,  and  we  later  studied  in  detail 
the  properties  of  such  antihemolytic  sera.  On  injecting  rabbits 
with  specific  hemolytic  serum  from  guinea-pigs  immunized  against 
rabbit  blood  we  found  that  these  rabbits  formed  an  antiserum 
that  would  inhibit  the  hemolytic  effect  of  the  specific  guinea-pig 
serum,  and  that,  strangely  enough,  acted  against  both  substances 
necessary  in  hemolysis.  In  other  words,  it  destroyed  the  charac- 
teristic sensitizer,  and  was  also  antitoxic  for  guinea-pig  alexin 
(anti-alexic  property).  On  account  of  its  anti-alexic  property  this 
antiserum  protected,  not  only  rabbit  corpuscles,  but  also  other  sen- 
sitized cells,  against  guinea-pig  alexin.  For  example,  it  was  found 
that  sensitized  cholera  vibrios  (treated  with  heated  cholera  serum) 
could  be  subjected  to  fresh  guinea-pig  serum  without  showing 
granular  disintegration,  provided  a  suitable  dose  of  antiserum  was 
added. f  In  short,  the  addition  of  antiserum  to  guinea-pig  alexin 
prevented  both  its  hemolytic  and  its  bacteriolytic  action.  The 
anti-alexin  was  found,  moreover,  to  be  strictly  specific,  having  no 
effect  on  the  hemolytic  or  bactericidal  action  of  sera  from  animals 
other  than  the  guinea-pig. 

The  anti-alexic  property  of  such  antisera  was  later  studied  by 
several  observers.  Wassermann  in  particular  confirmed  our 
experiment  on  the  antibactericidal  effect  of  anti-alexin  and  in 
addition  noted  an  interesting  modification.  He  neutralized  the 
alexin  of  normal  serum  by  anti-alexin  in  vivo  instead  of  in  vitro; 
he  found  that  he  could  annul  the  bactericidal  power  of  the  peritoneal 
exudate  by  injecting  antiserum  into  the  peritoneal  cavity. 

The  antisensitizing  property  has  recently  interested  other  experi- 
menters. In  the  present  article  we  shall  consider  the  facts  that 
have  been  brought  out  concerning  it. 

*  Agglutination  and  dissolution  of  red  blood  cells  by  serum,  p.  165. 

Hemolytic  sera,  their  antitoxins,  etc.,  p.  186. 

t  This  antiserum,  of  course,  had  been  deprived  of  its  own  alexin  by  heating  to  55 
degrees.  This  temperature  does  not  effect  the  anti-alexin  that  protects  the  vibrios. 


282  STUDIES  IN   IMMUNITY. 

I.    PROPERTIES  OF  ANTISENSITZERS. 

The  antisensitizer  should  be  chosen  with  some  care  in  order  to 
render  its  study  most  fruitful.  The  antisensitizing  power  is  fre- 
quently only  slightly  developed  in  antisera  and  is  often  detectable 
only  by  delicate  methods;  in  which  cases  very  large  doses  of  anti- 
serum  must  be  used  to  neutralize  the  sensitizer  effectively,  and  such 
doses  are  often  inconvenient  for  various  reasons. 

For  this  reason  one  must  use  an  antiserum  with  marked  antisen- 
sitizing properties  and  also  an  active  sensitizer.  It  is  necessary, 
too,  that  the  normal  sera  of  the  animals  furnishing  the  sensiti- 
zer and  the  antisensitizer  respectively  should  be  as  inactive  as  pos- 
sible, as  they  are  used  as  controls  to  contrast  with  the  peculiar 
properties  of  the  immune  sera.  These  conditions  are  very  satis- 
factorily realized  in  the  following  example: 

The  sensitizer  we  have"  generally  used  is  the  serum  of  rabbits 
that  have  been  given  three  or  four  injections  of  from  5  to  7  c.c. 
each  of  defibrinated  bovine  blood.  In  certain  other  experiments 
we  have  used  as  well  the  serum  of  rabbits  immunized  against  other 
blood  corpuscles,  for  example,  human  and  hen  corpuscles.  The 
antisensitizer  employed  is  the  serum  of  guinea-pigs  that  have 
received  two  or  three  injections  of  3  to  5  c.c.  each,  at  intervals  of 
from  12  to  15  days,  of  normal  rabbit  serum.  These  animals  are  bled 
2  weeks  after  the  last  injection. 

Before  being  used  for  experiments,  both  sera  are  deprived  of 
alexin  by  heating  for  a  half  hour  to  55  to  56°  C.  As  we  already 
know,  this  temperature  has  no  effect  on  the  sensitizer  or  antisensi- 
tizer. For  simplicity,  we  refer  to  our  sensitizers  as  "  rabbit  >  ox, 
56  degrees/'  " rabbit  >  hen,  56  degrees,"  " rabbit  >  human,  56 
degrees, "  and  the  antisensitizer  as  guinea-pig  >  rabbit  antiserum, 
56  degrees.  The  control  sera  are  normal  rabbit  serum,  56  degrees, 
normal  guinea-pig  serum,  56  degrees. 

What  is  the  best  method  of  demonstrating  antisensitizing  power? 
Two  widely  divergent  methods  may  be  considered. 

First,  sensitizer  and  antiserum  may  be  mixed,  the  corpuscles 
used  as  a  reagent  subsequently  added,  and  a  determination  made 
as  to  whether  they  have  become  sensitized  by  subsequently  adding 
alexin.  This  method  is  one  frequently  employed.  It  makes  use 


PROPERTIES   OF  ANTISENSITIZERS.  283 

of  the  antiserum  as  a  preventive,  the  sensitizer  being  neutralized 
before  it  has  affected  the  corpuscles. 

We  may  also  attempt  to  "cure"  corpuscles  already  treated  with 
the  sensitizer  by  means  of  the  antiserum.  Attempts  of  this  sort 
have  been  made  by  Pfeiffer  and  Friedberger,*  who  worked  with 
bacteria  instead  of  blood  corpuscles.  Such  experiments  are  also 
similar  to  those  of  Madsenf  and  Kraus  and  Lipschiitz,t  who  cured 
blood  cells  intoxicated  with  bacterial  poisons  by  means  of  antitoxins. 

This  second  method  of  experimentation  has  seemed  to  us  prefer- 
able. It  allows  one  to  treat  a  single  sensitizer  with  the  antiserum 
to  the  exclusion  of  other  sensitizers.  In  addition  to  the  specific 
sensitizer  in  rabbit  >  ox  serum  there  are  one  or,  according  to 
certain  authors,  a  large  number  of  similar  normal  sensitizers  already 
present  in  the  animal  before  immunization.  If  we  begin  by  mix- 
ing the  antiserum  with  rabbit  >  ox  serum,  it  is  probable  that  the 
normal  sensitizers  will  take  part  in  the  reaction.  But  if,  instead, 
we  first  mix  the  sensitizing  serum  with  the  corpuscles  and  then 
wash  them  to  remove  the  excess  of  serum,  and  add  them,  laden  as 
they  are  with  the  specific  substance,  to  antiserum,  the  latter  should 
affect  only  the  specific  sensitizer  united  with  the  blood  cells. 

It  remains  to  choose  a  suitable  alexin,  which  should  be  one  that 
is  not  destroyed  or  weakened  by  the  antiserum.  The  simplest  thing 
is  to  employ  fresh  guinea-pig  serum,  as  this  is  the  species  of  animal 
that  furnishes  the  antiserum.  Not  only  is  this  alexin  not  affected 
by  the  antiserum,  but  it  acts  very  well  in  conjunction  with  the 
sensitizers  employ ed.§ 

Following  is  the  method  of  demonstrating  antisensitizing  action : 

Sensitized  blood,  and  also  control  non-sensitized  blood,  are  first 
prepared.  In  each  of  two  large  tubes  is  placed  1  c.c.  of  washed 
bovine  blood.  ||  To  tube  A  is  then  added  2  c.c.  of  rabbit  >  ox 

*  Centralblatt  fur  Bakt.  Orig.,  XXXIV,  72. 

t  Zeitschrift  fur  Hygiene,  XXXII. 

J  Idem,  XLV,  49. 

§  Human  alexin  may  also  be  used. 

||  This  washed  blood  has  been  restored  to  its  original  volume,  that  is  to  say, 
contains  as  many  red  blood  cells  to  a  given  volume  as  did  the  original  blood.  A 
small  amount  of  defibrinated  blood  is  poured  into  a  tube  and  the  level  marked  on 
the  glass.  After  filling  with  salt  solution  and  centrifugalization,  the  supernatant 
fluid  is  removed  and  sufficient  salt  solution  added  to  restore  the  sediment  to  its 
original  volume. 


284  STUDIES   IN   IMMUNITY. 

serum,  56  degrees,  and  to  tube  B,  2  c.c.  of  normal  rabbit  serum, 
56  degrees.  Half  an  hour  later  the  two  tubes  are  filled  with  salt 
solution,  shaken  and  centrifugalized.  The  supernatant  fluids  are 
pipetted  off  and  the  sedimented  corpuscles  in  each  tube  are  sus- 
pended in  3  c.c.  of  salt  solution.  These  two  corpuscle  suspen- 
sions differ  only  in  that  the  first  corpuscles  are  laden  with  specific 
sensitizer.  We  may  then  prepare  the  following  mixtures : 

Tube  a.  Sensitized  blood,  0.1  c.c. ;  Guinea-pig  >  rabbit  antiserum,  56  degrees, 
0.3  c.c. 

Tube  b.  Same  as  last,  but  containing  0.3  c.c.  of  normal  guinea-pig  serum, 
56  degrees,  instead  of  antiserum. 

Tubes  c  and  d.  Same  as  "a"  and  "b"  respectively,  but  containing  non- 
sensitized  blood  in  place  of  sensitized  blood. 

One  hour  later  0.1  of  a  cubic  centimeter  of  guinea-pig  alexin  (fresh  serum)  is 
added  to  each  tube. 

It  is  found  that  hemolysis  takes  place  rapidly  in  tube  "b."  In 
tubes  "a,"  "c,"  and  "d"  there  is  no  hemolysis  even  after  24  hours. 
The  lack  of  hemolysis  in  "a"  is  due  to  the  neutralization  of  the 
sensitizer  by  the  antiserum,  as  may  be  shown  by  adding  a  small 
amount  of  rabbit  >  ox  serum,  56  degrees,  which  at  once  produces 
hemolysis.  The  same  results  are  obtained  if  we  use  any  other 
alexin  (for  example,  human  serum)  that  is  not  affected  by  the  anti- 
serum. 

This  method  of  experimentation  allows  us  to  measure  the  anti- 
sensitizing  power  of  the  antiserum.  We  may  add  in  place  of  0.3 
of  a  cubic  centimeter  of  pure  antiserum  to  0.1  of  a  cubic  centimeter 
of  sensitized  blood,  0.3  of  a  cubic  centimeter  of  a  dilution  of  anti- 
serum  in  a  greater  or  less  amount  of  heated  normal  guinea-pig 
serum.  On  trial  we  find  that  the  antisera  from  different  guinea- 
pigs  vary  much  in  potency.  The  antisensitizing  property  of  anti- 
serum  is  frequently  so  strong  that  0.1  of  a  cubic  centimeter  will 
suffice  to  protect  0.1  of  a  cubic  centimeter  of  sensitized  blood  from 
the  alexin. 

Having  settled  on  our  technic,  we  may  now  consider  certain 
questions  that  naturally  arise  concerning  antisensitizers :  Certain 
of  the  questions  (B,  C,  D  and  E)  relate  particularly  to  the  effect  of 
the  antiserum  on  the  sensitizer;  others  (A  and  F)  relate  rather  to 
the  origin  of  and  the  variations  in  antibodies. 

(A)     Is  an  antiserum  obtained  by  immunizing  an  animal  of 


PROPERTIES   OF  ANTISENSITIZERS,  285 

species  A  with  the  serum  of  normal  untreated  animals  of  species  B 
capable  of  neutralizing  the  specific  sensitizers  formed  by  species 
B  against  such  cells  as  red  blood  corpuscles?  Experimentally,  we 
learn  that  it  is;  for  our  antiserum,  which,  as  already  shown,  is 
actively  antisensitizing,  was  obtained  from  guinea-pigs  that  had 
received  only  normal  rabbit  serum.  The  same  results  are  true  if 
we  replace  the  sensitized  ox  blood  by  hen  or  human  blood,  each 
sensitized  by  its  respective  hemolytic  serum  from  the  rabbit  (rabbit 
>  hen,  56  degrees,  or  rabbit  >  human,  56  degrees).  This  agrees 
very  well  with  the  findings  of  various  writers  and  particularly  with 
those  of  Ford.*  This  writer,  having  found  that  hen  corpuscles  are 
agglutinated,  not  only  by  the  serum  of  a  rabbit  immunized  with 
hen  blood,  but  also  to  a  certain  extent  by  normal  rabbit  serum, 
injected  hens,  on  the  one  hand,  with  normal  rabbit  serum,  and  on 
the  other  with  the  specific  serum  of  rabbits  that  had  been  immunized 
against  hen  blood  (rabbit  >  hen  serum).  He  found  that  either 
antiserum  from  the  hen  neutralized  both  the  specific  agglutinin 
of  rabbit  >  hen  serum  and  the  normal  agglutinin  of  normal  rabbit 
serum.  Pfeiffer  and  Friedbergerj  obtained  analogous  results  with 
antisera  for  bacteria. 

We  may  conclude,  then,  from  these  results  that  as  a  general  rule 
antiserum  obtained  by  injecting  the  normal  serum  of  species  A,  and 
acting,  therefore,  on  the  normal  antibodies,  mill  also  neutralize  the 
various  specific  antibodies  formed  by  A  in  response  to  immunization. 

(B)  Is  the  antisensitizer  used  up  in  producing  its  effect  as  are 
the  other  antibodies  already  studied?  In  other  words,  is  not  the 
amount  of  sensitizer  that  an  antiserum  can  neutralize,  limited? 
It  is  almost  superfluous  to  add  that  this  turns  out  to  be  true ;  there 
is  a  minimal  dose  of  antiserum,  a  less  amount  than  which  fails  to 
protect  sensitized  corpuscles.  And,  moreover,  it  may  be  shown 
that  the  addition  of  a  sufficient  amount  of  washed  sensitized  bovine 
corpuscles  to  antiserum  deprives  it  of  its  antisensitizing  property, 
as  is  shown  on  adding  additional  sensitized  corpuscles  to  such 
treated  antiserum.  J  Of  course  antiserum  treated  with  the  same 

*  Zeit.  fur  Hygiene  XL,  1902,  363. 

f  Berliner  klin.  Woch.,  1902,  No.  1,  and  Centralblatt  fur  Bakt.,  XXXIV, 
1903,  74. 

J  We  shall  consider  this  experiment  in  detail  in  another  connection. 


286  STUDIES  IN  IMMUNITY. 

amount  of  non-sensitized  corpuscles  (i.e.,  treated  with  normal  rabbit 
serum,  56  degrees,  and  then  washed)  loses  no  antisensitizing  power. 

(C)  Does  the  antiserum  neutralize  the  sensitizer  directly,  like  a 
true  antitoxin,  or  does  it  simply  neutralize  the  effect  of  this  sub- 
stance in  some  antagonistic  manner?    To  answer  this  question  we 
must  determine  whether  corpuscles  sensitized  and  then  cured  by 
antiserum  remain  refractory  to  alexin  even  after  being  washed  in 
salt  solution. 

Experimentally,  we  find  that  they  do.  We  add  to  0.1  of  a  cubic 
centimeter  of  sensitized  blood  0.3  of  a  cubic  centimeter  of  anti- 
serum;  in  another  tube  we  add  to  a  similar  amount  of  sensitized 
blood  0.3  of  a  cubic  centimeter  of  heated  normal  guinea-pig  serum. 
After  a  certain  time  we  fill  both  tubes  with  salt  solution,  centrifu- 
galize,  decant  the  supernatant  fluid,*  and  suspend  the  sedimented 
corpuscles  in  0.3  of  a  cubic  centimeter  of  heated  normal  guinea-pig 
serum.  We  then  add  0.1  of  a  cubic  centimeter  of  guinea-pig  alexin 
to  each  tube.  There  is  no  hemolysis  in  the  tube  that  contained 
antiserum,  but  rapid  hemolysis  in  the  other.  The  curing  of  sen- 
sitized corpuscles,  therefore,  does  not  depend  on  permanent  contact 
with  the  antiserum. 

(D)  Does  the  antiserum  inhibit  the  effect  of  the  sensitizer  in 
respect  to  its  various  manifestations?    The  most  important  prop- 
erty of  the  sensitizer  is  to  render  the  suitable  cells  susceptible 
to  destruction  by  alexin.    But,  as  we  have  already  shown,  sensi- 
tizers  also  have  the  property  (a  property,  moreover,  correlative 
with  the  other)  of  conferring  on  the  specific  cells  the  power  of  absorb- 
ing alexin  and  of  so  removing  it  from  the  surrounding  fluid.     Con- 
sequently we  must  ascertain  whether  corpuscles  that  are  sensitized 
and  then  treated  with  antiserum  will  still  fix  alexin.     Experimentally, 
we  find  that  under  these  conditions  the  alexin  is  not  absorbed. 

Defibrinated  washed  ox  blood  is  mixed  with  either  two  volumes 
of  rabbit  >  ox  serum,  56  degrees,  or  of  normal  rabbit  serum,  56 
degrees.  After  sufficient  contact  the  tubes  are  filled  with  salt  solu- 
tion, centrifugalized,  and  the  supernatant  fluids  decanted.  One 
volume  of  salt  solution  is  added  to  each  blood  sediment.  As  a 
result  we  have  two  similar  emulsions  of  red  blood  corpuscles,  one  of 
which  is  sensitized.  There  are  then  prepared  the  following  tubes: 
*  This  washing  may  be  repeated  several  times. 


PROPERTIES  OF  ANTISENSITIZERS.  287 

Tube  a.  Sensitized  blood,  0.3  c.c.;  Normal  guinea-pig  serum,  56  degrees, 
1.2  c.c. 

Tube  b.  Same  as  last,  with  guinea-pig  >  rabbit  antiserum,  56  degrees,  replac- 
ing the  normal  serum. 

Tubes  c  and  d.  Same  as  "a"  and  "b"  respectively,  with  non-sensitized  blood 
in  place  of  sensitized  blood. 

One  hour  later  0.1  of  a  cubic  centimeter  of  alexin  (fresh  guinea- 
pig  serum)  is  added  to  each  tube.  Hemolysis  takes  place  in  tube 
"a"  in  a  few  minutes;  no  hemolysis  in  the  other  tubes.  The  tubes 
are  shaken  from  time  to  time  and  after  about  five  hours  are  cen- 
trifugalized.  The  clear  supernatant  fluids  are  placed  in  separate 
tubes,  and  0.4  of  a  cubic  centimeter  of  a  mixture  composed  of  one 
part  of  washed  ox  blood  to  two  parts  of  rabbit  >  ox  serum,  56 
degrees,  is  added  to  each  tube.* 

These  new  sensitized  corpuscles  are  rapidly  hemolyzed  in  all 
tubes  except  the  one  containing  supernatant  fluid  "a,"  where  no 
hemolysis  occurs,  f  We  conclude  from  this  experiment  that  the 
antiserum  removes  from  sensitized  corpuscles  their  power  of  fixing 
alexin;  such  corpuscles  act  as  do  normal  corpuscles. 

(E)  Does  the  antisensitizer  drive  out  the  sensitizer  from  the 
corpuscles  by  a  process  of  washing,  or  does  it  unite  with  the  sensitizer 
joined  to  the  corpuscle? 

We  have  just  seen  that  if  a  sufficient  amount  of  sensitized  cor- 
puscles is  added  to  the  antiserum  the  latter  becomes  inactive. 
We  might  suppose  that  the  antisensitizer  drives  out  the  sensitizer 
from  the  corpuscles,  and  the  fact  that  it  becomes  inert  might  be 
due  to  a  mutual  saturation  of  the  two  antagonistic  substances. 

Let  us  take  0.1  of  a  cubic  centimeter  of  sensitized  ox  blood  and 
add  to  it  0.3  of  a  cubic  centimeter  of  antiserum.  After  a  little 
contact  we  wash  the  corpuscles  carefully  in  salt  solution.  After 
centrifugalization  and  decanting  we  suspend  the  sedimented  cor- 
puscles in  0.3  of  a  cubic  centimeter  of  an  active  normal  guinea-pig 
serum,  and  add  0.1  of  a  cubic  centimeter  of  alexin.  No  hemolysis 
occurs.  Has  the  sensitizer  with  which  the  corpuscles  were  laden 
been  driven  out  by  the  antiserum  and  then  removed  from  the  fluid 

*  The  sensitizing  serum  in  this  mixture  is  relatively  so  large  in  amount  that 
no  neutralization  of  it  could  take  place  in  tube  "b,"  even  if  the  antisensitizer  had 
not  been  used  up. 

t  Hemolysis  will  take  place  in  this  tube  on  addition  of  a  little  more  alexin. 


288  STUDIES  IN  IMMUNITY. 

by  washing?  To  settle  this  question  we  add  0.1  of  a  cubic  cen- 
timeter of  normal  rabbit  serum,  56  degrees.*  On  this  addition 
hemolysis  soon  appears.  In  control  tubes  we  find  that  this 
normal  rabbit  serum  has  in  itself  no  power  to  sensitize  ox 
corpuscles.  We  must  therefore  conclude  that  the  sensitizer  has 
remained  with  the  corpuscles,  that  the  antisensitizer  has  joined 
with  it,  but  has  not  expelled  it,  and  that  normal  rabbit  serum 
contains  a  substance  (normal  sensitizer?)  that  is  able  to  break  up 
the  combination  of  specific  sensitizer  and  antisensitizer  by  re- 
placing the  first  in  its  union  with  the  second  substance.  And  if 
normal  rabbit  serum  under  these  conditions  seems  to  sensitize,  it 
does  so  indirectly  by  liberating  the  specific  sensitizer  that  has  been 
neutralized. 

We  shall  later  return  to  this  experiment  in  other  connections, 
particularly  in  considering  the  multiplicity  of  active  substances 
in  a  given  immune  serum.  We  may  note  simply,  for  the  moment, 
that  this  phenomenon,  which  may  be  designated  as  a  suppression 
by  normal  serum  of  a  cure  effected  by  antiserum,  varies  in  rapidity 
with  the  conditions  of  the  experiment.  We  have  already  remarked 
that  the  reappearance  of  sensitization  on  adding  normal  rabbit 
serum  becomes  slower  and  more  difficult  in  proportion  to  the  time 
of  contact  between  the  corpuscle  and  the  antiserum  before  the 
addition  of  the  normal  serum.  In  other  words,  it  would  seem,  so 
far  as  our  experiments  go,  that  the  combination  between  specific 
sensitizer  and  antisensitizer  becomes  more  perfect  in  some  way 
with  time  and  less  apt  to  be  affected  by  the  sensitizers  of  normal 
serum.  The  combination  would  also  seem  to  be  more  stable  when 
the  antiserum  employed  is  very  powerful. 

It  is  not  surprising  that  normal  rabbit  serum  should  contain 
substances  with  an  affinity  for  the  antisensitizer.  We  showed  in 
1899  that  substances  similar  to  sensitizers  f  may  be  detected  in 
normal  sera,  and  Ehrlich  and  Morgenroth  have  furnished  numerous 
analogous  examples.  In  consideration  of  the  experiment  we  have 

*  It  may  be  remembered  that  our  antiserum  was  obtained  by  injecting  guinea- 
pigs  with  normal  rabbit  serum. 

t  Although  these  substances  would  seem  to  belong  to  the  same  category  as 
sensitizers,  they  are,  with  few  exceptions,  very  inferior  to  them  as  regards  their 
affinity  for  the  sensitive  cells.  They  are,  moreover,  very  little  known,  and  we  call 
them  "normal  sensitizers,"  only  with  a  certain  reservation. 


PROPERTIES   OF   ANTISENSITIZERS.  289 

just  discussed,  it  would  seem  superfluous  to  add  that  if  normal 
rabbit  serum,  56  degrees,  is  added  in  the  first  place  to  the  anti- 
serum  the  latter  loses  its  power  to  protect  sensitive  corpuscles 
from  alexin.  It  is  indeed  obvious,  since  even  when  the  corpuscles 
have  been  cured  by  antiserum  the  subsequent  addition  of  normal 
rabbit  serum  will  neutralize  the  protection  already  afforded. 

Another  fact  resulting  from  the  evidence  of  the  preceding  experi- 
ment is  that  sensitized  ox  corpuscles  cured  with  antiserum  and 
washed,  and  resistant  to  guinea-pig  or  human  alexin,  will  be  hemo- 
lyzed  by  rabbit  alexin,  since  this  serum  restores  the  sensitization. 

Is  the  power  of  annulling  the  cure  of  corpuscles  by  antiserum 
present  in  normal  rabbit  serum  that  has  been  heated  to  tempera- 
tures considerably  above  56  degrees?  Heating  to  70  degrees  for  a 
half  hour  will  not  destroy  it.  On  the  other  hand,  the  fluid  ex- 
pressed from  a  clot  of  serum  coagulated  at  100  degrees  no  longer 
retains  this  power.  The  power  is  only  feebly,  if  at  all,  present  in 
rabbit  aqueous  humor  heated  to  56  degrees.  It  is  evident,  then, 
that  if  it  be  due  to  normal  sensitizers,  as  is  likely,  they  are  not 
present  in  the  aqueous  humor.  This  fact  is  in  harmony  with  our 
experimental  results  published  in  1895,  in  which  we  demonstrated 
that  the  bactericidal  power  of  cholera  serum  is  due  to  the  collabora- 
tion of  two  distinct  substances,  one  a  specific  one  which  is  ther- 
mostable and  occurs  only  in  the  serum  of  vaccinated  animals 
(preventive  substance  or  sensitizer) ;  and  the  other,  the  alexin,  or 
proper  bactericidal  substance,  destroyed  at  55  degrees,  and  present 
in  the  serum  of  both  normal  and  vaccinated  animals  in  approxi- 
mately equal  amounts.  The  familiar  experiment  of  mixing  the 
aqueous  humor  (56  degrees)  of  an  immunized  animal  with  fresh 
serum  of  a  normal  animal  and  vibrios  gave  no  bacteriolysis;  bac- 
teriolysis was  energetic,  however,  if  heated  serum  from  the  same 
animal  replaced  the  aqueous  humor.  In  other  words,  aqueous 
humor  contains  no  sensitizer. 

(F)  If  we  find  that  a  given  antiserum  neutralizes,  as  does  the 
one  we  are  studying,  several  different  specific  sensitizers,  each 
active  against  different  cells,  but  all  derived  by  immunizing  animals 
of  the  same  species,  are  we  to  conclude  that  this  antiserum  contains 
several  different  antisensitizers,  each  one  of  which  combines  with 
a  separate  sensitizer?  Or  are  we  to  conclude  that  the  antiserum 


290  STUDIES  IN   IMMUNITY. 

contains  a  single  antisensitizer  that  neutralizes  various  sensitizers 
indifferently? 

Although  this  question  has  not  been  put  to  experimental  proof, 
it  would  seem  to  be  answered  by  certain  results  on  another  problem 
considered  by  Wassermann  *  and  by  Ford.f  These  investigators 
have  Uied  to  elucidate  the  following  point :  In  many  instances,  as 
we  know,  the  serum  of  a  normal  animal  has  a  distinct  agglutinating 
power,  without  any  immunization,  for  blood  corpuscles  A,  let  us 
say. I  If  this  animal  is  immunized  against  corpuscle  A,  is  the 
specific  agglutinin  that  is  formed  to  be  considered  as  identical  with 
the  one  present  in  the  normal  serum  before  treatment?  In  brief, 
does  immunization  simply  increase  a  preexisting  principle  already 
present  in  small  amount,  without  giving  rise  to  new  and  particular 
substances,  properly  speaking?  If  this  is  so,  immunization  would 
be  equivalent  simply  to  a  purely  quantitative  modification. 

Wassermann  and  Ford  think  that  the  fact  brought  out  by  Ford, 
to  which  reference  has  been  made,§  makes  clear  this  obscure  point 
and  proves  conclusively  that  antibodies  active  against  a  given  cell 
in  normal  or  in  immune  serum  are  identical.  The  fact,  to  repeat, 
is  that  the  antiserum  from  hens  immunized  against  normal  rabbit 
serum  neutralizes  the  agglutinating  effect  of  either  normal  rabbit 
serum  or  of  the  serum  of  rabbits  immunized  against  hen  corpuscles 
for  the  corpuscles  in  question.  Their  conclusion  is  as  follows: 
if  the  antiserum  obtained  by  injection  of  normal  agglutinin  neutral- 
izes both  normal  and  immune  agglutinin,  it  proves  that  these  agglu- 
tinins  are  identical. 

We  do  not  see  why  Wassermann  and  Ford  consider  this  con- 
clusion logical.  It  would  be  true  only  if  it  had  been  proved  that  a 
given  anti-agglutinin  (or  antisensitizer)  could  under  no  circum- 
stances neutralize  several  different  agglutinins  (or  sensitizers). 
And  this  is  precisely  what  Wassermann  and  Ford  have  not  proved. 
Why,  therefore,  if  wo  were  to  formulate  in  the  beginning  the  opposite 

*  Wassermann,  Zeitschrift  fUr  Hygiene,  XLII,  1903,  267. 

t  Ford,  Zeitschrift  fUr  Hygiene,  XL,  1902,  363. 

J  Wassermann  and  Ford  deal,  it  is  true,  with  agglutinins  and  not  with  sensi- 
tizers. It  is  not,  however,  unreasonable  to  apply  the  results  obtained  with 
agglutinins  to  sensitizers  and  consequently  conclusions  concerning  anti-agglu- 
tinins  to  antisensitizers. 

§  See  p.  285. 


PROPERTIES   OF   ANTISENSITIZERS.  291 

hypothesis  —  namely,  that  one  and  the  same  anti-agglutinin  (or 
antisensitizer)  will  neutralize  indifferently  the  various  agglutinins 
formed  by  a  given  animal  species — should  we  not  be  justified  in 
asserting  that  the  two  agglutinins  in  question,  both  affecting  hen 
blood  corpuscles,  are  not  identical,  but  show  distinct  differences  as 
marked,  let  us  assume,  as  those  between  immune  agglutinins  from 
the  same  animal  species  affecting  different  cells?  Why,  in  fact, 
should  we  accept  without  experimental  proof  the  thesis  that 
Wassermann  and  Ford  consider  axiomatic,  that  there  is  a  specific 
antibody  exclusively  fitted  for  each  active  substance  and  that,  con- 
versely, when  two  substances  are  neutralized  by  a  given  antibody 
that  they  are  identical?  Why,  in  short,  must  we  conclude  from 
Ford's  experiment  that  immunization  simply  increases  the  amount 
of  the  active  substance  without  changing  it  qualitatively?  It  is 
evidently  indispensable  to  consider  this  question  before  we  can 
admit  Wassermann  and  Ford's  conclusion,  the  importance  of  which 
in  increasing  our  comprehension  of  the  genesis  of  active  substances 
in  immune  sera  is  evident.  We  shall  see  from  the  following  experi- 
ments, as  a  matter  of  fact,  that  a  given  antisensitizer  can  neu- 
tralize distinctly  different  sensitizers  affecting  different  cells. 

When  we  mix  our  antiserum  with  sensitized  and  washed  ox 
corpuscles  the  antisensitizer  is,  as  we  know,  used  up  in  curing  the 
corpuscles,  so  that  the  supernatant  fluid  after  centrifugalization 
no  longer  protects  sensitized  blood  corpuscles.  But  will  this  fluid 
still  protect  other  corpuscles  sensitized  with  a  different  rabbit  sen- 
sitizer,  for  instance,  hen  corpuscles  treated  with  rabbit  >  hen 
serum?  The  experiment,  the  details  of  which  follow,  proves  that 
it  will  not.  The  same  antisensitizer  neutralizes,  then,  two  different 
sensitizers  (rabbit  >  ox  and  rabbit  >  hen).  In  a  control  it  is 
shown  that  ox  corpuscles  treated  with  rabbit  >  hen  serum  (which 
they  do  not  absorb)  will  not  remove  from  the  antiserum  the  power 
of  protecting  sensitized  hen  or  ox  corpuscles.  In  the  same  way 
it  is  shown  that  rabbit  >  ox  serum  does  not  sensitize  hen  cor- 
puscles. 

Washed  ox  blood,  the  volume  of  which  equals  the  primitive 
blood,  is  placed  in  equal  amounts  in  two  large  tubes  (1.5  c.c.)  and 
two  volumes  (3  c.c.)  of  rabbit  >  ox  serum,  56  degrees  added  to  one, 
and  the  same  amount  of  rabbit  >  hen  serum,  56  degrees,  to  the 


292  STUDIES  IN  IMMUNITY. 

other.  After  contact  the  corpuscles  are  carefully  washed,  centrifu- 
galized,  and  the  supernatant  fluids  decanted;  1.5  c.c.  of  salt  solu- 
tion is  then  added  to  each  tube.  In  one  tube,  then,  the  blood  is 
sensitized  and  in  the  other  it  is  not. 

In  the  same  way  hen  blood,  sensitized  and  not  sensitized,  is 
prepared  by  treating  it  with  each  specific  serum. 

Antiserum  is  then  diluted  with  an  equal  volume  of  normal  guinea- 
pig  serum,  56  degrees,  to  facilitate  mensuration.  We  refer  to  this 
as  diluted  antiserum.*  We  then  prepare  the  following  mixtures 
of  antiserum  and  the  two  ox  bloods  treated  as  described: 

Tube  A.     Sensitized  ox  blood,  1  c.c.;  diluted  antiserum,  2.5  c.c. 

Tube  B.     Same  as  A,  with  non-sensitized  ox  blood  in  place  of  sensitized  blood. 

One  hour  later  the  tubes  are  centrifugalized  and  the  supernatant  fluids  A  and 
B  decanted.  Fluid  A  is  deprived  of  antisensitizer,  as  may  be  imagined.  These 
fluids  are  used  in  making  the  following  tubes: 

Tubes  C  and  D.  Each  contains  1  c.c.  of  fluid  A;  to  C  is  added  0.1  of  a  cubic 
centimeter  of  sensitized  ox  blood.  To  D  is  added  0.1  of  a  cubic  centimeter  of 
sensitized  hen  blood. 

Tubes  E  and  F.  The  same  as  tubes  C  and  D  respectively,  with  fluid  B  replacing 
fluid  A. 

In  addition  there  are  controls  containing  each  1  c.c.  of  normal  guinea-pig  serum, 
56  degrees,  and  0.1  of  a  cubic  centimeter  of  hen  or  ox  blood  sensitized  and  not 
sensitized  respectively.  These  control  the  tubes  containing  antiserum. 

Three-quarters  of  an  hour  later  0.2  of  a  cubic  centimeter  of  alexin  (fresh 
guinea-pig  serum)  is  added  to  tubes  C,  D,  E,  F,  and  to  the  controls. 

There  is  no  hemolysis  in  tubes  E  and  F  containing  liquid  B,  in 
which  the  antiserum  has  not  been  deprived  of  its  antisensitizing 
power  by  sensitized  corpuscles.  There  is,  of  course,  no  hemolysis 
in  the  controls  with  non-sensitized  corpuscles  and  without  anti- 
serum.  In  tubes  C  and  D  that  contain  fluid  A,  in  which  the 
antisensitizer  has  been  previously  used  up  by  the  sensitized  ox  cor- 
puscles, the  hemolysis  of  both  the  ox  and  the  hen  corpuscles  takes 
place  as  rapidly  as  in  the  controls  without  antiserum.  The  same 
antisensitizer,  then,  neutralizes  the  two  sensitizers  under  con- 
sideration, which  differ  markedly,  as  one  is  specifically  suitable 
for  ox  corpuscles  and  combines  with  them,  whereas  the  other 
does  not. 

*  It  may  be  noted  that  four  parts  of  this  antiserum  completely  protect  one 
part  of  the  sensitized  blood  used  against  the  subsequent  action  of  alexin.  In 
a  control  tube  in  which  the  antiserum  is  replaced  by  normal  guinea-pig  serum, 
56  degrees,  hemolysis  is  complete  in  5  minutes. 


PROPERTIES   OF  ANTISENSITIZERS.  293 

This  experiment  is  not  indispensable,  as  a  similar  conclusion 
might  be  drawn  with  assurance  from  an  observation  that  has  been 
made  concerning  normal  rabbit  serum.  We  have  already  noted 
that  on  adding  a  little  normal  rabbit  serum,  56  degrees,  to  a  mix- 
ture of  sensitized  corpuscles,  antiserum  and  alexin,  in  which  no 
hemolysis  is  present,  hemolysis  appears;  we  must  conclude,  then, 
that  the  normal  serum  contains  a  substance  that  can  replace  the 
specific  sensitizer  in  its  union  with  the  antitoxin,  at  least  in  part. 
That  this  substance  is  not  to  be  regarded  as  a  rabbit  >  ox  sensi- 
tizer preexistent  in  small  amounts  in  normal  rabbit  serum  is  evident 
from  the  fact  that  the  normal  serum  does  not  sensitize  ox  cor- 
puscles to  alexin.  But  in  order  to  be  quite  certain  of  this  point 
and  to  forestall  any  possible  objection,  we  may  ascertain  whether 
normal  rabbit  serum  that  has  been  treated  with  an  excess  of  ox 
corpuscles  acts  in  a  similar  manner. 

Let  us  place  1  c.c.  of  defibrinated  ox  blood  in  a  large  tube,  fill  with 
salt  solution,  centrifugalize,  and  then  remove  the  supernatant  fluid, 
leaving  a  sediment  of  blood  corpuscles  to  which  1  c.c.  of  heated 
rabbit  serum  is  added.  On  the  following  day  we  centrifugalize, 
remove  the  supernatant  serum  and  add  to  it  the  fresh  sediment 
of  another  cubic  centimeter  of  washed  ox  blood.  We  may  hope 
that  two  successive  contacts  with  ox  corpuscles  will  have  absorbed 
all  the  combinable  substances  in  the  normal  serum.  We  then 
prepare  sensitized  and  washed  ox  blood  as  in  the  former  experi- 
ment. 

Two  tubes,  A  and  B,  are  prepared,  containing,  each,  0.1  of  a  cubic 
centimeter  of  sensitized  washed  blood,  0.3  of  a  cubic  centimeter  of 
antiserum  and  0.1  of  a  cubic  centimeter  of  guinea-pig  alexin;  to 
tube  A  is  then  added  0.2  of  a  cubic  centimeter  of  the  normal  rabbit 
serum  treated  with  ox  blood  as  described.  The  corpuscles  are 
hemolyzed  in  10  minutes  in  tube  A,  but  remain  intact  in  tube  B. 
A  control  shows  that  the  treated  normal  serum  in  the  same  doses 
does  not  hemolyze  a  mixture  of  antiserum,  alexin  and  non-sensi- 
tized corpuscles. 

The  same  results  are  obtained  by  using  in  place  of  normal  rabbit 
serum,  rabbit  >  ox  serum  that  has  previously  been  deprived  of 
its  specific  activity  by  two  successive  contacts  with  ox  corpuscles. 
Such  treated  rabbit  >  ox  serum  still  combines  with  the  anti- 


294  STUDIES  IN   IMMUNITY. 

sensitizer,  although  deprived  of  all  its  specific  sensitizing  property 
for  ox  blood ,  or,  in  other  words,  when  it  contains  only  normal  sensi- 
tizers  with  no  particular  affinity  for  ox  corpuscles.* 

We  are  not  justified  in  concluding,  therefore,  as  Wassermann  and 
Ford  do,  that  two  sensitizers  (or  agglutinins)  are  identical  simply 
because  they  are  neutralized  by  the  same  antibody.  The  con- 
clusion of  these  observers  concerning  the  identity  of  two  agglutinins 
affecting  the  same  blood  cells,  and  of  which  one  is  present  in  normal 
sera  and  the  other  is  produced  by  immunization,  may  not  reason- 
ably be  drawn  from  their  experiments.  It  may  be  exact,  but  has 
not  yet  been  proved. 

The  answer  to  question  F  is  that  a  single  given  antisensitizer  can 
neutralize  several  different  sensitizers  from  the  same  animal  species, 
but  active  for  different  cells.  When  we  come  to  consider  the  fact 
noted  by  other  observers  (Ehrlich  and  Morgenroth,  Pfeiffer  and 
Friedberger),  namely,  that  an  antiserum  obtained  by  injecting  an 
animal  of  species  A  with  the  serum  of  species  B  has  little  or  no 
effect  on  sensitizers  from  either  species  C  or  D,  we  must  conclude 
that,  as  far  as  sensitizer  action  is  concerned,  there  is  a  closer  rela- 
tion between  sensitizers  from  a  common  source  active  against  different 
cells  than  there  is  between  sensitizers  active  against  the  same  cell  and 
obtained  from  different  animal  species. 

II.     OBSERVATIONS  ON  THE  CHEMICAL  THEORIES  OF  IMMUNITY. 

The  study  of  antisensitizers  suggests  various  remarks  of  which 
note  should  be  made;  these  remarks,  indeed,  would  seem  to  facili- 
tate the  comprehension  of  certain  insufficiently  explained  or 
inaccurately  interpreted  already  known  facts.  We  shall  consider, 
therefore,  first  of  all,  Ehrlich's  theory  and  later  the  mechanism  of 
passive  immunity. 

There  is  yet  another  subject  which  may  be  considered  more 
attentively,  and  that  is  the  mode  of  action  of  toxins  on  antitoxins, 
toward  the  elucidation  of  which  the  study  of  antisensitizers  would 
seem  to  offer  interesting  information.  Indeed,  owing  to  the 

*  We  may  repeat  that,  in  designating  these  substances  as  normal  sensitizers, 
we  simply  mean  that  they  have  the  power,  as  do  specific  sensitizers,  of  combining 
with  antisensitizer.  We  know,  however,  relatively  little  of  their  nature  and  prop- 
erties. We  give  them  a  definite  name  simply  to  facilitate  expression. 


PROPERTIES   OF   ANTISENSITIZERS.  295 

property  that  blood  cells  have  of  extracting  their  specific  sensitizer 
from  heated  hemolytic  serum,  we  are  able  to  simplify  the  toxic  solu- 
tion, or,  in  other  words,  to  isolate  the  toxin  to  be  tested  with  the 
antitoxin.  And  the  fact  that  the  normal  sensitizers  of  normal 
rabbit  serum  (which  are  not  toxic  for  the  ox  corpuscles,  but  have 
as  great  an  affinity  for  the  antitoxin  as  the  specific  sensitizer),  when 
added  to  neutralized  toxin  (i.e.,  sensitized  corpuscles  +  antiserum), 
liberate  it  by  uniting  with  a  certain  amount  of  antitoxin,  may  allow 
us  to  determine  the  law  that  governs  the  equilibrium  between 
antagonistic  substances.  But  we  shall  not  discuss  in  the  present 
article  the  theories  of  the  interaction  of  toxins  and  antitoxins,  as  our 
researches  on  this  subject  are  not  yet  finished.  We  may  simply 
mention  a  fact  that  would  seem  to  indicate  that  the  destruction 
of  toxin  by  antitoxin  is  rarely  absolute — whether  because  the  re- 
action is  incomplete,  so  that  the  mixture  always  contains  traces 
of  free  toxin  (Arrhenius  and  Madsen),  or  because,  as  we  believe,  the 
neutralization  of  a  toxin  by  an  antitoxin  is,  in  reality,  simply  a 
greater  or  less  attenuation  depending  on  whether  the  toxin  fixes 
more  or  less  antitoxin.  According  to  our  hypothesis  the  two 
substances  unite  in  variable  properties  and  may  therefore  give 
rise  to  a  series  of  combinations  ranging  from  free  toxin  to  com- 
pletely saturated  toxin,  being  less  and  less  toxic  (without  becoming, 
of  necessity,  quite  non-toxic)  in  proportion  to  the  increase  in  anti- 
toxin. As  we  know,  Ehrlich's  toxons,  which  occur  in  mixing  a 
toxin  with  an  incompletely  neutralizing  dose  of  antitoxin,  represent 
in  Arrhenius  and  Madsen's  conception  small  amounts  of  free  toxin; 
according  to  our  conception  they  are  toxic  molecules  insufficiently 
saturated  with  antitoxin  in  which  the  toxicity  is  simply  decreased 
without  being  eliminated.  It  would  seem  to  us,  then,  that  in  mix- 
ing antiserum  and  sensitizer  we  form  toxons  from  the  toxin,  in  that 
the  sensitizer,  when  affected  by  antiserum,  is  simply  weakened,  but 
not  deprived  of  its  original  power.  The  sensitization  is,  as  we  know, 
very  slight,  so  that  under  the  ordinary  conditions  of  experimentation, 
that  is  to  say,  in  a  mixture  containing  serum,  the  red  blood  cells 
remain  intact.  The  sensitizing  power  has  not,  however,  been 
entirely  destroyed,  for  we  find  evidence  of  it  if  the  corpuscles  are 
made  less  vulnerable  by  being  placed  in  a  less  favorable  medium 
(salt  solution) ;  in  other  words,  under  conditions  that  facilitate  sen- 


296  STUDIES  IN  IMMUNITY. 

sitization.  Under  these  conditions  the  antiserum  is  unable  to 
prevent  hemolysis. 

Let  us  add  0.6  of  a  cubic  centimeter  of  antiserum,  which  is  a 
large  dose,  to  0.2  of  a  cubic  centimeter  of  sensitized  ox  blood.  We 
fill  the  tube  with  salt  solution,  centrifugalize,  and  remove  the  super- 
natant fluid.  To  the  sediment  we  add  0.6  of  a  cubic  centimeter  of 
normal  guinea-pig  serum,  56  degrees,  and  0.2  of  a  cubic  centimeter  of 
guinea-pig  alexin.  Under  these  conditions  no  hemolysis  occurs. 
The  experiment  is  repeated,  with  the  variation  of  adding  0.6  of  a 
cubic  centimeter  of  normal  salt  solution  in  place  of  the  heated 
guinea-pig  serum  after  washing  and  then  adding  the  alexin  (0.2 
c.c.).  Hemolysis  occurs  completely  in  about  an  hour.*  The  cure 
of  the  corpuscles,  then,  was  only  partial  and  the  sensitization,  al- 
though weakened,  is  evident  when  the  corpuscles  are  placed  in  a 
medium  that  lowers  their  resistance.! 

Are  we  not  justified  in  comparing  this  fact  with  the  one  noted 
some  time  ago  by  Roux  and  Vaillard,  namely,  that  a  mixture  of 
tetanus  toxin  and  antitoxin  that  is  harmless  for  normal  guinea-pigs 
is  dangerous  for  guinea-pigs  that  have  been  weakened  by  vacci- 
nation with  the  cholera  vibrio? 

Without  considering  these  questions  further  for  the  moment, 
we  may  return  to  the  subject  properly  under  consideration. 

Ehrlich's  theory. — Relates  to  the  origin  of  antibodies  and  is  as 
follows:  on  injecting  an  animal  with  a  substance  that  gives  rise  to 
an  antibody,  the  substance  injected  unites  with  certain  chemical 
elements  (receptors)  in  certain  definite  cells.  These  cells  are  dis- 
turbed and  react.  In  reestablishing  their  previous  condition  they 
form  new  receptors  which,  being  produced  in  excess,  are  forced  out 
of  the  cell  into  the  surrounding  fluids  and  constitute  the  anti- 
bodies. 

The  theory  offers,  then,  an  easy  means  for  determining  the  nature 
of  antibodies.  Let  us  apply  it  to  the  antisensitizer  we  have  been 
studying.  When  guinea-pigs  are  immunized  against  rabbit  serum 
we  must  conceive  of  them  as  having,  according  to  the  theory, 

*  A  control  shows  that  the  same  corpuscles  without  sensitization  are  unaffected. 
We  know,  furthermore,  that  heated  normal  guinea-pig  serum  has  no  antisensitiz- 
ing  effect. 

f  It  is  a  well-known  fact  that  salt  solution  renders  corpuscles  less  resistant  to 
traces  of  hemolytic  serum. 


PROPERTIES   OF  ANTISENSITIZERS.  297 

receptors  capable  of  combination  with  certain  active  principles 
(normal  sensitizers)  of  normal  rabbit  serum.  The  receptors  affected 
by  the  injection  are  reproduced  in  excess  by  a  cellular  reaction  and 
poured  into  the  serum,  which  fluid  becomes  endowed  with  the 
property  of  neutralizing  the  specific  rabbit  >  ox  sensitizer.  Con- 
sequently, the  antisensitizer  is  composed  of  receptors  identical 
with,  or  similar  to,  the  receptors  in  the  ox  corpuscles  that  unite 
with  the  sensitizer.*  It  should  then  act  as  these  corpuscle  recep- 
tors do,  for  it  is,  so  to  speak,  only  a  solution  of  such  receptors  in 
serum.  This  conception  of  the  antisensitizer  is  indeed  the  one 
recently  accepted  by  Morgenroth.f 

The  inaccuracy  of  such  a  conception  founded  on  Ehrlich's  theory 
is  evident  from  the  facts  we  have  just  offered.  In  the  first  place, 
if  the  antisensitizer  were  identical  with  the  corpuscle  receptors 
used,  it  would  not  combine  with  the  sensitizer  already  saturated 
with  these  receptors,  in  other  words,  bound  to  the  corpuscles.  And 
the  cure  of  sensitized  cells  by  antiserum  would  be  impossible. 

Moreover,  if  the  antisensitizer  were  identical  with  the  receptors 
in  question,  it  is  evident  that  any  substance  which  would  combine 
with  one  would  combine  with  the  other.  Normal  rabbit  serum  con- 
tains, as  we  know,  no  substances  that  combine  with  the  receptors 
of  ox  corpuscles  (that  is,  these  corpuscles  remove  nothing  from  the 
serum),  and  yet  this  serum  is  so  avid  of  antisensitizer  that  it 
can  compete  successfully  with  rabbit  >  ox  sensitizer  in  combining 
with  it.  Rabbit  >  ox  serum,  moreover,  even  when  entirely  de- 
prived of  its  specific  sensitizer  for  ox  corpuscles,  will  still  saturate 
antisensitizer.  And,  what  is  more,  since  the  same  antisensitizer 
unites  with  various  sensitizers  indifferently,  whether  or  not  they 
combine  with  ox  corpuscles  the  result  is  that  antiserum  treated  with 
sensitized  ox  corpuscles  no  longer  protects  other  varieties  of  sen- 
sitized corpuscles. 

The  theory  under  discussion  is  also  in  direct  opposition  to  the 
fact  that  antiserum  removes  from  sensitized  corpuscles  their  power 
of  absorbing  alexin.  It  is  evident  that  if  the  antisensitizer  were 
made  from  corpuscle  receptors,  its  combination  with  the  sensitizer 

*  According  to  Ehrlich's  terminology,  these  receptors  all  have  the  same  hapto- 
phore  group. 

f  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  and  Sons,  p.  241. 


298  STUDIES  IN  IMMUNITY. 

should  take  up  alexin  energetically  in  the  same  way  as  sensitizer 
plus  corpuscle  does.  This  is  not  found  to  be  true.* 

To  sum  up:  first,  the  antisensitizer  unites  with  the  sensitizer 
already  fixed  on  the  specific  corpuscles,  which  latter  it  cures;  second, 
the  antiserum  owes  its  activity  to  a  single  unique  substance  that 
endows  it  with  the  property  of  protecting  various  sensitized  cells; 
in  other  words,  of  neutralizing  various  sensitizers  that  combine  each 
with  a  different  type  of  receptor;  for  example,  the  same  antisensi- 
tizer combines  not  only  with  rabbit  >  ox  sensitizer,  but  also  with 
other  normal  or  specific  sensitizers  that  do  not  unite  with  ox  cor- 
puscles; third,  the  antisensitizer  removes  from  sensitized  corpuscles 
their  power  of  absorbing  alexin.  All  the  facts  appear  to  be  irrecon- 
cilable with  Ehrlich's  theory,  and  tend  to  prove  the  general  thesis 
that  antitoxins  (or  other  antibodies)  are  not  assimilated  by  the  recep- 
tors that  fix  their  respective  toxins.  We  consider  them  all,  whether 
acting  against  bacterial,  vegetable  or  animal"  toxins,  as  substances 
of  the  same  nature  and  of  a  common  cellular  origin,  in  the  same  gen- 
eral category  and  with  marked  similarity.  If  the  receptor  theory 
were  true,  the  various  antitoxins  would  be  united  by  no  analogy, 
since  it  would  seem  reasonable  that  the  receptors  attacked  by 
various  toxins  must  be  widely  different,  according  to  the  nature 
and  property  of  the  poison  in  question. 

Ehrlich  and  Morgenroth  have  given  over  the  larger  part  of  one 
memoir  to  a  consideration  of  the  antisensitizers,f  the  importance 
of  which  is  essential  to  partisans  of  the  lateral-chain  theory.  This 
article,  indeed,  is  one  of  those  that  have  most  contributed  to  make 
this  conception  acceptable  and  to  offer  the  most  convincing  argu- 
ments of  its  accuracy.  The  facts  brought  forth  in  this  article  are 
in  direct  opposition  to  our  own  and  must,  therefore,  be  considered. 
We  shall  first  of  all,  however,  deal  with  an  article  recently  published 
by  Morgenroth  4 

*  If  we  were  to  accept  the  ideas  of  the  Ehrlich  school,  and  particularly  the 
ideas  that  the  sensitizer  combines  with  the  alexin  and  that  each  property  mani- 
fested by  a  substance  is  evidenced  by  a  particular  grouping  in  the  molecule,  one 
might  say  that  the  antisensitizer  should  unite  only  with  the  cytophilic  group  of 
the  sensitizer  and  not  with  its  complementophilic  group.  There  is  no  experi- 
mental evidence  for  this. 

t  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  and  Sons,  p.  88. 

t  Morgenroth,  Komplementablenkung  durch  hamolytische  Ambozeptoren. 
Centralblatt  fur  Bakt.,  XXXV,  1904,  501. 


PROPERTIES   OF  ANTISENSITIZERS.  299 

The  hypothesis  of  " complement  deviation"  (Komplementablen- 
kung)  formulated  to  explain  the  observations  of  Neisser  and  Wechs- 
berg  on  the  inhibiting  influence  of  too  large  an  amount  of  sensitizer 
on  bacteriolysis  when  the  amount  of  alexin  is  relatively  small,  is 
well  known.*  It  has  been  claimed,  without  direct  demonstration 
that,  since  the  bacteria  in  such  an  experiment  cannot  absorb  the 
large  amount  of  sensitizer,  the  excess  remaining  in  the  fluid  unites 
with  the  alexin  and  monopolizes  a  larger  or  smaller  part  of  it,  so 
that  it  does  not  attack  the  bacteria.  It  is  rather  strange  that  the 
inhibiting  effect  of  an  excess  of  sensitizer  has  never  been  noted  in 
hemolytic  experiments,  and  Morgenroth  has  attempted  to  fill  this 
rather  important  gap  in  the  theory.  He  admits  in  the  first  place 
that  hemotoxic  sensitizers  must  first  be  combined  with  the  recep- 
tors of  the  appropriate  corpuscles  in  order  to  show  any  marked 
affinity  for  the  alexin. f  Consequently,  in  a  mixture  of  corpuscles, 
alexin,  and  too  large  a  dose  of  sensitizer,  the  excess  of  the  latter 
substance  remaining  free  in  the  fluid  cannot  take  up  the  alexin 
because  it  needs  corpuscle  receptors  in  order  to  become  avid  of  the 
complement.  The  introduction  of  these  receptors  is  necessary  to 
produce  complement  deviation.  On  the  supposition  that  the 
antisensitizer  is  identical  with  corpuscle  receptors  as  far  as  affinities 
are  concerned  (since  they  both  possess  the  same  haptophore  group, 
according  to  the  lateral-chain  theory),  Morgenroth  conceived  the 
idea  that  a  mixture  of  sensitizer  and  antisensitizer  should  be  able 
to  fix  a  certain  amount  of  alexin.  Such  a  mixture  should  act,  in 
other  words,  precisely  as  we  have  shown  J  that  sensitizer  and  cor- 
puscles do,  that  is,  should  absorb  alexin  energetically.  It  is  evident 
that  if  these  two  substances  —  corpuscle  receptors  and  antisen- 
sitizer —  are  considered  as  identical  the  addition  of  either  one  of 
them  to  a  mixture  of  sensitizer  and  alexin  should  bring  about  the 
fixation  of  a  certain  amount  of  the  active  substance  and  conse- 

*  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  and  Sons,  p.  120.  See 
also  in  this  connection  this  volume,  p.  357. 

f  It  may  well  be  questioned  why  antimicrobial  sensitizers  should  not  be  subject 
to  the  same  necessity.  It  is  certain  that  if  the  experiment  had  turned  out  the 
other  way,  that  is  to  say,  if  the  phenomenon  of  complement  deviation  had  been 
found,  not  in  bacteriolysis,  but  in  hemolysis,  that  the  same  explanation  would  have 
been  forthcoming;  it  would  simply  have  been  necessary  to  apply  it  to  the  other 
instance. 

$  See  p.  191. 


300  STUDIES   IN   IMMUNITY. 

quently  diminish  the  hemolytic  power  of  the  fluid.  Sensitized 
corpuscles,  therefore,  used  as  a  reagent  for  the  destructive  power, 
would  be  less  likely  to  be  hemolzyed  when  added  to  a  mixture  of 
alexin,  antisensitizer  and  sensitizer,  than  when  added  to  a  mixture 
of  antisensitizer  and  alexin  without  sensitizer.  This  difference 
would  be  due  to  the  fact  that  in  the  first  instance  part  of  the  alexin 
would  be  consumed  by  the  complex  "sensitizer-antisensitizer," 
which  acts  precisely  as  does  "sensitizer-corpuscle,"  since,  according 
to  Morgenroth,  the  terms  antisensitizer  and  corpuscle  receptor  are 
synonymous. 

Experiment  confirms  Morgenroth's  expectations.  By  mixing, 
in  carefully  chosen  proportions,  guinea-pig  alexin,  rabbit  >  ox 
sensitizer  (56  degrees)  and  goat  >  rabbit  serum  (56  degrees)  it  may 
be  shown  that  the  alexin  does  not  remain  free.  If  sensitized  cor- 
puscles are  subsequently  added  to  such  a  mixture  and  at  the  same 
time  to  another  similar  mixture  without  the  sensitizer,  hemolysis 
appears  in  the  second,  but  not  in  the  first. 

Morgenroth,  however,  does  not  prove  that  the  results  are  really 
due  to  the  substance  which  he  holds  responsible  for  them.  There 
is  no  proof  that  the  disappearance  of  alexin  is  due  to  its  combina- 
tion with  the  rabbit  >  ox  sensitizer  united  to  the  receptors  of  anti- 
sensitizer. To  prove  that  it  is  this  sensitizer  that  uses  up  the 
alexin,  a  control  should  have  been  made  to  show  that  the  same 
result  is  not  obtained  in  a  mixture  of  alexin,  antisensitizer,  and 
an  immune  serum  that  has  been  already  deprived  of  its  specific 
sensitizer.* 

It  seems  to  us  that  Morgenroth's  phenomenon  should  be  explained 
differently.  In  addition  to  the  alexin  there  are  two  sera  in  the 
experiment  to  be  considered.  The  first  (antisensitizer)  has  been 
obtained  by  injecting  goats  with  the  second  serum,  namely,  of 
rabbits  immunized  with  ox  corpuscles.  The  first  is  certainly  anti- 
sensitizing  for  the  second  in  the  sense  that  it  neutralizes  its 
specific  sensitizer.  But  from  another  standpoint  it  may  be  re- 
garded as  sensitizing  for  the  same  serum,  and  this  fact  Morgen- 
roth does  not  take  into  account.  The  antiserum  (serum  I)  is 
from  animals  injected  with  alien  serum  (serum  II),  and  in  this 
respect  should  sensitize  serum  II  if  we  consider  this  serum  simply 
*  Or  in  a  mixture  of  alexin,  antisensitizer  and  normal  rabbit  serum. 


PROPERTIES   OF  ANTISENSITIZERS.  301 

as  a  solution  of  albuminous  substances,  precisely  as  the  serum 
of  animals  vaccinated  with  milk  sensitizes  milk;  in  other  words, 
confers  on  certain  of  the  constituents  of  milk  a  power  to  ab- 
sorb alexin.*  Gengou  has  demonstrated  the  interesting  fact  that 
"  anti-albuminous  sensitizers/'  similar  to  antimicrobial  or  anti- 
hematic  sensitizers,  may  be  obtained,  which  have  a  similar  power  of 
fixing  alexin  when  united  with  the  antigen.  Gengou  proved  the 
existence  of  such  sensitizers,  not  only  in  the  serum  of  animals  im- 
munized against  milk,  but  also  in  the  serum  of  animals  treated 
with  an  alien  serum.  Such  an  antiserum  plays  an  obvious  role 
in  Morgenroth's  experiments.  We  are  justified  in  supposing,  then, 
that  the  alexin  absorption  is  brought  about,  not  by  a  union  of  anti- 
sensitizer  with  sensitizer,  but  simply  by  certain  sensitized  albumin- 
oids of  the  rabbit  >  ox  serum. 

Morgenroth's  conclusion,  therefore,  is  not  acceptable  in  the 
present  state  of  our  knowledge.  As  far  as  we  are  concerned,  the 
theory  of  complement  deviation  by  amboceptor  (sensitizer)  is  a 
myth.  We  have  already  stated  that  an  identity  of  receptors  with 
antibodies  cannot  be  admitted.  It  is  not  only  incompatible  with 
our  own  results,  but  receives  no  experimental  confirmation  from 
the  work  of  Pfeiffer  and  Friedberger,  who  are  zealous  upholders  of 
Ehrlich's  theory. 

We  may  note  in  passing  that  Morgenroth  has  not  noticed  in  his 
experiments  that  his  antisensitizer  can  cure  already  sensitized  cor- 
puscles. In  this  his  result  differs  from  our  own;  but  we  used  a 
different  antiserum.  The  question,  however,  properly  arises  as  to 
whether  the  lavish  use  of  normal  salt  solution  (which,  as  we  have 
seen,  tends  to  annul  corpuscle  protection  to  a  great  extent  and 
should  therefore  be,  as  much  as  possible,  eliminated  from  hemolytic 
experiments  f)  has  not  affected  the  accuracy  of  this  author's  obser- 
vations. 

Let  us  now  consider  as  briefly  as  possible  Ehrlich  and  Morgen- 
roth's ideas  on  antisensitizers  as  stated  in  their  sixth  memoir  on 
hemolysis. 

These  authors  employ  an  immune  serum  from  rabbits  immu- 
nized against  ox  blood.  They  find  that  this  serum  gives  hemolysis 
with  various  alexins  and  particularly  with  those  of  the  guinea-pig 
*  See  Gengou,  p.  241.  f  See  also  Gay,  p.  333. 


302  STUDIES  IN   IMMUNITY 

and  the  goat.  They  find,  however,  that  when  they  use  goat  alexin 
the  corpuscles  must  be  more  heavily  sensitized  (that  is,  more  heated 
immune  serum  added)  than  when  they  use  guinea-pig  alexin.  The 
fact,  however,  is  not  surprising.  It  is  well  known  that  the  alexins 
from  different  animals  are  not  strictly  identical;  and,  since  they 
differ  somewhat,  it  would  be  strange  indeed  if  they  had  equal  hemo- 
lytic  power  for  a  given  blood  cell ,  and  it  is  not  astonishing  that  the 
corpuscles  have  to  be  made  more  vulnerable  by  a  heavier  sensitiza- 
tion  in  order  for  some  alexins  to  destroy  them,  whereas  a  weak 
sensitization  suffices  with  other  alexins.  Ehrlich  and  Morgenroth, 
however,  reject  so  simple  an  explanation.  They  think,  rather,  that 
the  immune  serum  contains  two  (or  more)  distinct  sensitizers,  "x" 
and  "y";  the  one  in  greatest  abundance  (x)  is  very  efficient  with 
the  guinea-pig  alexin,  but  finds  no  fit  complement  in  the  goat  serum; 
this  first  sensitizer,  then,  has  nothing  to  do  with  an  hemolysis  caused 
by  goat  alexin.  This  alexin,  however,  suits  the  second  sensitizer 
(y)  of  the  serum  very  well,  but,  since  this  "y  "  is  present  in  relatively 
small  amounts,  a  large  amount  of  immune  serum  must  be  used  when 
goat  alexin  is  employed.  Let  us  accept,  for  the  sake  of  argument, 
this  idea  of  the  multiplicity  of  sensitizers  in  a  given  immune  serum 
in  spite  of  its  improbability,  and  go  on  to  the  next  point. 

Ehrlich  and  Morgenroth  have  a  second  serum,  an  antiserum 
from  goats  obtained  by  immunizing  them  with  rabbit  >  ox  immune 
serum,  which  latter  serum  it  neutralizes.  First  of  all,  they  add  to 
a  large  dose  of  this  antiserum  (Dose  A,  let  us  say)  a  small  amount 
of  rabbit  >  ox  serum  and  then  add  ox  corpuscles.  These  corpus- 
cles are  then  washed  and  guinea-pig  alexin  added  to  them.  There 
is  no  hemolysis,  showing  that  the  sensitizing  power  has  been  abol- 
ished by  the  antiserum.  Their  conclusion  is  as  follows :  The  anti- 
serum  is  antitoxic  for  sensitizer  "x"  suitable  for  guinea-pig  alexin. 

Ehrlich  and  Morgenroth  then  perform  a  second  similar  experi- 
ment, using  in  this  instance  goat  alexin.  Under  these  conditions 
the  antiserum  apparently  does  not  protect  the  corpuscles  and 
hemolysis  occurs.  This  is  the  conclusion:  The  antisensitizer  is 
so  specific  that,  although  it  can  neutralize  sensitizer  "x,"  it  has  no 
effect  on  sensitizer  "y,"  which  is  particularly  suited  to  goat  alexin. 
This  fact  goes  to  show  that  there  are  indeed  two  distinct  sensitizers. 

Their  second  experiment,  however,  differs  from  the  first  in  an 


PROPERTIES  OF  ANTISENSITIZERS.  303 

important  detail.  Imbued  with  the  idea  that  the  rabbit  >  ox 
immune  serum  contains  only  a  small  amount  of  "y"  suitable  for 
goat  alexin,  Ehrlich  and  Morgenroth  think  it  indispensable  to  mix 
with  the  given  dose  A  of  antiserum,  as,  in  the  first  experiment,  a 
much  larger  amount  of  rabbit  >  ox  immune  serum  than  was  used 
in  the  first  instance.  They  think  this  technic  is  justified  because 
even  a  large  amount  of  the  serum  would  contain  only  a  small 
amount  of  the  "y"  which  is  to  be  tested  against  the  antiserum  and 
is  alone  of  importance,  since  it  is  the  only  sensitizer  that  can  hemo- 
lyze  the  corpuscles  in  conjunction  with  goat  alexin.  They  do  not 
consider  that,  in  adding  this  excess  of  immune  serum,  they  introduce, 
not  only  a  large  amount  of  specific  sensitizer,  which  is  the  only 
substance  they  have  in  mind,  but  also  a  large  amount  of  normal 
sensitizers,  which,  as  we  know,  monopolize  a  greater  part  of  the  anti- 
sensitizing  power  and  so  prevent  the  specific  sensitization  from  being 
neutralized  by  the  antiserum.  Of  course  under  these  conditions 
the  corpuscles  are  hemolyzed.  A  small  amount  of  normal  serum 
would  have  been  just  as  effective  in  hiding  the  antisensitizing 
effect  as  was  the  excess  of  immune  serum.  Under  such  conditions 
no  corpuscle  protection  would  have  been  evident  even  if  guinea- 
pig  alexin  were  used  instead  of  goat  alexin.  Ehrlich  and  Morgen- 
roth's  laborious  considerations  on  the  nature  of  antisensitizers, 
on  the  multiplicity  of  antibodies,  and  particularly  on  the  multi- 
plicity of  sensitizers  in  a  given  immune  serum  lead  to  an  incorrect 
conception  of  the  experimental  results.  It  is  evident  from  this 
example  that  the  logic  employed  in  defending  the  lateral-chain 
theory  is  far  from  unassailable;  indeed,  in  certain  instances  it  is 
open  to  severe  criticism  and  cannot  be  accepted  without  argument. 
Theories  of  passive  immunity.  —  The  immunity  conferred  by 
injecting  preventive  serum  presents  certain  peculiarities  that  have 
recently  attracted  considerable  attention.  It  was  formerly  thought 
that  the  animal  that  received  antitoxin  and  benefited  from  it 
acted  simply  as  a  passive  recipient  without  any  reaction,  or  the 
elaboration  of  any  antagonistic  substance  even  when  the  serum 
injected  was  from  an  alien  species.  The  fact  that  the  duration 
of  passive  immunity  is  brief  was  always  supposed  to  be  due  to 
a  gradual  elimination  of  the  antibodies.  This  was  the  general 
opinion,  which  we  ourselves  shared  when  we  demonstrated  in  1895 


304  STUDIES  IN   IMMUNITY. 

that  the  collaboration  of  two  substances  was  necessary  for  the 
bacteriolysis  of  the  cholera  vibrio.  We  were  able  to  satisfy  our- 
selves that  an  animal  immunized  with  cholera  serum,  and  thereby 
acquiring,  as  Fraenkel  and  Sobernheim  showed,  a  bacteriolytic 
power  in  their  body  fluids,  owes  this  newly  obtained  power  to, 
first,  the  alexin  already  present  in  the  normal  animal,  and,  secondly, 
the  specific  sensitizer  in  the  injected  cholera  serum.  The  bac- 
tericidal power,  then,  is  generated  in  the  fluids  of  the  treated  animal 
as  it  is  in  a  test  tube  containing  fresh  normal  serum  on  the  addition 
of  a  little  anticholera  sensitizer.  In  either  instance  a  combination 
of  the  two  substances  is  required.*  The  fact  that  the  immunity 
soon  disappears  is  due  simply  to  disappearance  of  the  sensitizer 
from  the  fluids. 

This  explanation  of  the  transitory  nature  of  passive  immunity 
is  doubtless  correct  in  those  instances  in  which  the  injected  serum 
comes  from  animals  of  the  same  species.  But  it  has  been  noted 
by  several  observers  that  the  duration  of  the  immunity  is  remark- 
ably short  when  the  serum  is  obtained  from  an  alien  species.t 
Pfeiffer  and  Friedberger  offered  the  plausible  hypothesis  in  the 
case  of  cholera  serum  that,  when  passive  immunity  disappears  very 
rapidly,  it  is  owing  to  the  fact  that  the  treated  animal  reacts  to  the 
substances  injected  by  elaborating  antagonistic  substances,  like 
antisensitizers,  that  neutralize  them. 

The  question  may  well  arise  as  to  whether  animals  of  species  A 
inoculated  with  any  immune  serum  from  species  B  (antitoxins, 
agglutinins,  lactoserum  and  so  forth)  do  not  form  antisubstances 
to  them,  comparable  to  those  that  neutralize  hemolytic  sensitizers. 
As  we  have  noted,  Pfeiffer  and  Friedberger  obtained  an  anticholera 
serum  and  SchiitzeJ  has  obtained  an  antilactoserum.  Kraus  and 
Eisenberg§  have  made  systematic  studies  of  these  substances  and 
failed  to  find  in  the  serum  of  treated  rabbits  substances  capable 
of  neutralizing  the  diphtheria  antitoxin  or  the  typhoid  agglutinins 
present  in  specific  sera  from  the  horse.  Such  results  appear  irrecon- 

*  See  p.  80. 

f  See  particularly  among  recent  articles,  Schiitze,  Ueber  das  Verschwinden 
verschiedener  Immunsera  aus  dera  tierischen  Organismus.  Festsch.  v  60  Geburt- 
stag.  v.  R.  Koch,  p.  657. 

J  Schutze,  Berlin,  klin.  Wochen.,  1901,  No.  50,  1263. 

§  Kraus  and  Eisenberg,  Cent,  f .  Bakt.  Orig.  XXXI,  1902,  208. 


PROPERTIES   OF  ANTISENSITIZERS.  305 

cilable.     Why  is  not  the  law  that  controls  these  phenomena  more 
general? 

To  explain  the  contradictory  facts  in  harmony  with  Ehrlich's 
theory  the  following  explanation  has  been  offered:  Diphtheria 
antitoxin  and  typhoid  agglutinins  have  affinity  for  only  such 
receptors  as  occur  in  their  respective  micro-organisms.  It  is  natural, 
then,  that  these  substances  should  remain  free  in  the  injected  animal, 
since  they  find  no  appropriate  receptors;  as  these  receptors  are 
lacking,  they  are  not  reproduced  and  consequently  no  active  anti- 
serum  is  formed.  It  might  be  objected  that  if  this  explanation 
were  true  that  Pfeiffer  and  Fried  berger  would  have  obtained  no 
antisensitizer  to  cholera  serum.  But  this  objection  could,  in  turn, 
be  answered  by  saying  that  although  the  injected  animal' has  no 
receptors  identical  with  those  of  typhoid  or  diphtheria  bacilli 
and  consequently  fitted  to  fix  the  active  substances  that  affect 
these  organisms,  they  do  possess  receptors  similar  to  those  of  the 
cholera  vibrio.  If  the  experiment  had  turned  out  the  other  way, 
that  is  to  say,  if  an  antityphoid  serum  had  been  obtained  more 
easily  than  an  anticholera  serum,  the  converse  explanation  of  the 
respective  absence  or  presence  of  receptors  could  have  been  offered. 
In  short,  whether  an  antiserum  is  obtained  or  not,  the  receptor 
theory  is  upheld;  in  unfavorable  cases  receptors  are  lacking;  in 
favorable  cases  they  are  present.  Whatever  happens,  the  use  of 
the  term  "receptors"  makes  the  facts  easily  explicable. 

Another  explanation  of  these  divergent  results  may,  however,  be 
offered.  As  we  have  already  seen,  the  serum  of  a  guinea-pig  im- 
munized against  rabbit  serum  neutralizes  the  various  specific 
sensitizers  from  the  rabbit  indifferently,  and  also  the  normal  sen- 
sitizers  (or  substances  of  this  nature)  in  normal  rabbit  serum.  And, 
what  is  more,  a  single  given  antisensitizer  confers  all  these  antagonis- 
tic powers  on  the  antiserum  or  is  able,  in  other  words,  to  show  these 
various  affinities  by  uniting  with  the  various  sensitizers. 

It  follows,  therefore,  that  to  neutralize  a  given  specific  sensitizer 
(e.g.,  rabbit  >  ox  sensitizer)  economically  by  means  of  an  anti- 
serum  it  is  well  to  have  the  sensitizer  in  a  relatively  pure  condition, 
without  admixture  of  other  sensitizers,  before  treating  it  with- the 
antiserum.  Under  these  conditions  the  neutralizing  effect  of 
the  antiserum  is  directed  only  against  the  sensitizer  in  question. 


306  STUDIES  IN  IMMUNITY. 

This  purification  of  the  sensitizer  is  accomplished  in  our  experi- 
ments in  which  we  have  used  corpuscles  treated  with  their  specific 
serum.*  If  this  precaution  is  neglected  by  mixing  the  whole 
immune  serum,  which  contains  other  analogous  substances  in 
addition  to  the  specific  sensitizer,  with  the  antiserum  in  the  first 
place,  the  neutralizing  effect  is  wasted.  When  we  add  to  our 
sensitized  corpuscles,  either  before  or  after  mixing  them  with  anti- 
serum,  normal  sensitizers  in  the  form  of  normal  rabbit  serum,  56 
degrees  (or  indeed  rabbit  >  ox  serum  deprived  of  its  specific  sen- 
sitizer by  contact  with  ox  corpuscles)  their  cure  is  gravely  com- 
promised, since  the  neutralizing  power  of  the  antiserum  ceases 
to  be  concentrated  on  the  sensitizer  affecting  the  corpuscles.  If, 
therefore,  the  mixture  is  made  in  the  usual  manner,  antiserum  and 
whole  immune  serum  containing  the  specific  sensitizer  (or  anti- 
toxin or  agglutinin,  for  these  remarks  are  apparently  applicable 
to  any  of  the  antibodies)  to  be  neutralized,  the  chance  of  obtaining 
a  neutralization  depends  on  the  relation  of  specific  and  normal  sen- 
sitizer content  in  the  immune  serum.  The  greater  the  relative  amount 
of  normal  sensitizers  the  less  the  specific  sensitizer  will  be  affected 
by  the  antiserum,  so  that  its  neutralization  may  be  practically  nil. 
And  if  the  antisensitizers — or,  one  may  say,  the  an ti -anti toxins— 
are  found  to  be  of  relatively  little  potency  or  practically  negative, 
it  is  due  at  least  in  part  to  the  fact  that  not  all  their  activity  is  evi- 
dent. When  we  expect  to  neutralize  a  given  immune  body  in  an 
immune  serum  we  do  not  consider  that  energy  may  be  exhausted  in 
saturating  other  substances  that  are  not  taken  into  account  in  the 
experiment  and  of  the  presence  which  we  may  actually  be  ignorant. 
It  would  be  strange,  indeed,  that  the  relative  proportion  of  normal 
and  specific  sensitizers  should  be  the  same  in  all  immune  sera  from 
no  matter  what  animal  species.  It  must  be  expected,  then,  that 

*  It  would  be  desirable,  to  be  sure,  to  obtain  pure  sensitizer  in  some  other  way. 
It  would  then  be  possible  to  treat  it  with  antiserum  before  it  was  united  with  the 
blood  cells.  Hitherto  such  a  separation  has  not  been  possible,  although  it  would 
be  theoretically  better  than  the  method  we  have  employed.  It  is  indeed  possible 
that  the  antisensitizer  should  show  no  preventive  action  on  pure  sensitizer, 
although  it  can  cure  sensitized  corpuscles.  It  would  seem,  indeed,  from  our 
experiments  that  there  is  some  struggle  between  the  affinity  of  the  sensitizer  for 
the  corpuscle  on  the  one  hand,  and  for  the  antisensitizer  on  the  other.  And  it 
may  be  that  in  certain  cases  the  combination  between  sensitizer  and  corpuscle 
may  be  so  stable  as  to  prevent  any  curative  action  by  an  antiserum. 


PROPERTIES   OF  ANTISENSITIZERS.  307 

anti-antibodies  are  not  always  detectable  experimentally,  which 
may  account  in  part  for  the  variation  in  results.  There  are  different 
affinities  to  be  considered  in  these  experiments.  There  is  the 
affinity  of  the  anti-antibody  for  the  specific  antibody  and  for 
the  normal  antibody,  and  then  there  is  the  affinity  of  the  antibody 
for  the  sensitive  cell  used  as  reagent.  The  susceptibility  of  this 
reacting  cell  must  also  be  taken  into  account.  The  net  result  from 
these  factors  varies,  and  yet  is  the  only  result  that  we  take  into 
consideration.  The  laws  that  govern  these  phenomena  may  in 
reality  be  general,  although  the  apparent  results  differ.  The  study 
of  immunity  is  full  of  such  instances.  It  is  a  general  rule  that  the 
injection  of  bacteria  gives  rise  to  sensitizers,  and  these  sensitizers 
increase  the  destructive  power  of  the  alexin.  It  is  exceptional, 
nevertheless,  that  the  immune  serum  obtained  in  this  way  is  strongly 
bactericidal  for  the  specific  bacterium;  and  why?  It  is  due  to  the 
fact  that,  although  there  are  certain  bacteria  that  are  so  delicate 
as  to  be  destroyed  by  immune  sera,  the  majority  of  them  are  resist- 
ant and  suffer  no  injury  from  the  serum  in  a  suitable  medium,  as 
Metchnikoff  has  shown  by  numerous  examples. 

When  we  inject  animals  with  immune  serum  in  the  hope  of 
obtaining  an  anti-antibody  we  naturally  think  of  the  animal  as 
reacting  particularly  to  the  antibody  that  we  are  studying.  If 
the  experiment  succeeds,  as  is  the  case  with  cholera  serum,  and  if 
we  believe  in  Ehrlich's  theory,  we  conclude  that  the  animal 
has  formed  an  anticholera  serum  owing  to  the  reproduction  of 
receptors  analogous  to  those  of  the  cholera  vibrio.  But  this  con- 
ception does  not  represent  the  true  state  of  affairs.  If  the  cholera 
sensitizer  is  neutralized,  it  is  not  on  account  of  its  specific 
peculiar  qualities  and  simply  because  it  affects  the  cholera  vibrio. 
If  it  is  neutralized  it  is  not  owing  to  any  " personal  equation,"  so 
to  speak,  but  simply  because  it  belongs  in  common  with  all  sensi- 
tizers (and  probably  also  all  antitoxins)  to  a  group  of  substances 
belonging  to  species  A,  which,  on  injection  into  species  B,  cause  a 
reaction.  To  speak  colloquially,  we  might  say  that  the  injected 
animal  is  not  concerned  as  to  whether  it  is  given  antibodies  that 
affect  tetanus  or  diphtheria  toxin,  or  those  acting  on  cholera  or 
typhoid  bacilli ;  haptophore  groups  of  toxins  and  receptors  of  bacilli 
do  not  particularly  appeal  to  it. 


308  STUDIES  IN   IMMUNITY. 

Antibodies  belong  simply  to  a  group  of  active  substances  in  sera. 
The  only  affair  of  the  treated  animal  is  to  react  against  certain  or 
all  members  of  this  group,  representing,  as  they  do,  an  alien  sub- 
stance. Nor  must  it  be  lost  sight  of  that  in  injecting  immune 
serum  we  inject,  not  only  specific  antibody,  but  normal  serum  as  well. 
It  is  simply  the  unaccustomed  presence  of  all  these  foreign  sub- 
stances that  leads  the  animal  to  form  an  antagonistic  substance, 
and  as  a  result  this  substance  is  not  strictly  specific.  Such  an 
antiserum  may  be  used  to  protect  bacteria  or  various  kinds  of 
blood  cells  against  the  respective  antibodies  obtained  from  the 
same  animal  species.  Since  the  antibodies  obtained  by  immunizing 
with  normal  serum  will  neutralize  any  antibody  from  the  animal 
species  corresponding  to  the  normal  serum  employed,  it  is 
obvious  that  it  acts  on  the  entire  category  of  substances  in  this 
serum. 

Theoretically,  at  least,  we  might  expect  that  an  animal  would 
react  more  violently  on  injection  of  toxic  immune  serum  specific 
for  its  own  blood  cells.*  This  condition,  however,  is  not  indis- 
pensable in  order  to  produce  a  reaction,  as  is  reasonable  when 
we  consider  that  normal  serum  frequently  has  a  harmful  effect  on 
animals  of  a  different  species. 

When,  however,  an  animal  is  injected  with  a  specific  immune 
serum  acting  on  bacteria  or  cells  that  have  nothing  in  common  with 
the  vaccinated  animal,  and  as  a  result  an  antiserum  is  formed  that 
neutralizes  the  injected  antibody,  there  is  no  legitimate  reason 
for  us  to  conclude  that  there  is  any  necessary  identity  or  even  relation 
in  the  composition  of  this  animal's  cells  and  the  substances  (bac- 
teria, cells,  or  bacterial  products)  against  which  the  serum  in- 
jected is  specific.  There  is  no  reason  for  introducing  the  con- 
ception of  common  receptors.  As  a  matter  of  fact  the  antiserum 
obtained  under  these  conditions  does  not  differ  from  that 
obtained  on  injecting  normal  serum;  we  repeat  that  it  acts  indis- 
criminatingly  on  any  of  the  active  substances  present  in  the  foreign 
serum. 

We  believe,  then,  that  the  rapid  disappearance  of  passive  immunity 

*  It  may  be  noted,  however,  that  Kraus  and  Eisenberg  did  not  obtain  any 
detectable  active  antiserum  on  injecting  dogs  with  an  immune  serum  active  for 
dog  corpuscles. 


PROPERTIES  OF  ANTISENSITIZERS.  ek 

afforded  by  the  injection  of  an  alien  serum  should  be  attributed,  as 
a  rule,  to  the  secretion  of  antagonistic  substances.  The  fact  that 
this  substance  is  not  easily  detectable  experimentally  is  due  to  its 
inhibition  by  certain  of  the  conditions  to  which  reference  has  been 
made.  Such  conditions  depend  on  the  doses  used  and  the  relations 
of  affinity  between  the  substances  that  take  part  in  the  reaction. 
In  so  far  as  dosage  is  concerned,  it  should  be  noted  that  the  amount 
of  immune  serum  administered  for  passive  immunity  is  very  small 
in  proportion  to  the  volume  of  blood  in  the  recipient;  under  such 
conditions  the  antagonistic  power  that  soon  develops  has  greater 
chance  to  manifest  itself  than  in  test-tube  experiments,  where  the 
tendency  is  to  minimize  the  amount  of  antiserum. 

CONCLUSIONS. 

The  study  of  an  antiserum  obtained  by  injecting  animals  of  species 
A  with  the  normal  serum  of  species  B  gives  rise  to  the  following 
remarks : 

I.  Various  red  blood  cells,  sensitized  each  by  its  appropriate 
heated  hemolytic  serum  obtained    from  an  animal  of  species  B, 
lose  their  sensitization  to  alexin  when  treated  with  the  antiserum. 
The  sensitization  is  generally  diminished  rather  than  completely 
abolished;  it  may,  indeed,  frequently  be  demonstrated  when  the 
corpuscles  are  placed  in  a  medium  which  tends  to  diminish  their 
resistance,  for  example  in  salt  solution. 

II.  It  is  not  necessary  in  obtaining  an  antiserum  that  neu- 
tralizes various  specific  sensitizers,  obtained  in  each  instance  from 
an  animal  of  species  B,  to  inject  animals  with  the  respective  specific 
sensitizers,  but  simply  with  normal  serum  from  species  B. 

III.  The  power  of  this  antiserum  to  neutralize  various  specific 
sensitizers  as  well  as  the  normal  antibodies  (or  sensitizers)  in  B 
serum  may  be  attributed  to  the  presence  in  this  antiserum  of  a 
single  antisensitizer.    There  is  no  need  of  assuming  the  existence 
of  a  multiplicity  of  antisensitizers.    As  far  as  the  action  of  anti- 
sensitizer is  concerned,  there  is  a  closer  relation  between  sensitizers 
from  the  same  source  acting  on  different  cells  than  between  sen- 
sitizers from  different  sources  acting  on  the  same  cell. 

IV.  The  antisensitizer  is  used  up  in  acting.    The  addition  of  sen- 


310  STUDIES  IN   IMMUNITY. 

sitized  corpuscles  to  antiserum  removes  from  it  the  power  of  pro- 
tecting additional  sensitized  corpuscles  either  of  the  same  or  of  a 
different  variety.  The  normal  sensitizers  in  serum  B  also  neu- 
tralize the  antiserum. 

V.  The  antiserum  cures  sensitized  corpuscles  by  uniting  with 
the  sensitizer  that  is  fixed  on  them.    Corpuscles  protected  in  this 
way  resist  alexic  activity  even  when  the  excess  of  protective  serum 
has  been  washed  away. 

VI.  It  would  seem  as  if  the  complex  antisensitizer-sensitizer- 
red  blood  cell  were  broken  up  on  adding  normal  sensitizers  of 
serum  B  (i.e.,  normal  serum  or  immune  serum  deprived  of  specific 
sensitizers  by  contact  with  blood  cells),  because  these  sensitizers 
take  away  at  least  a  part  of  the  antisensitizer  that  has  already  been 
combined  with  the  specific  sensitizer. 

VII.  The  power  in  normal  serum  B  of  inhibiting  the  curative 
effect  of   the  antiserum   for  sensitized  corpuscles  resists  heating 
to  70  degrees,  but  not  to  100°  C.     It  is  not  present  in  aqueous 
humor  from  species  B.    It  has  previously  been  ascertained  that 
the  aqueous  humor  of  vaccinated  animals  contains  no  specific  sen- 
sitizer. 

VIII.  When  antiserum  affects  the  sensitizer  attached  to  red 
blood  cells  it  removes  from  the  complex  the  property  of  fixing  alexin. 

IX.  The  identity  of  the  antibodies  of  normal  and  of  immune 
serum  affecting  the  same  cell,  as  assumed  by  certain  writers,  must 
be  considered  as  not  proved. 

X.  Ehrlich's   theory,  which  supposes   that  specific  antibodies 
are  identical  with  the  cell  receptors  that  combine  with  those  sub- 
stances against  which  the  animal  becomes  immunized,  is  erroneous. 
The  arguments  to  support  this  theory  obtained  from  the  study  of 
antisensitizers  are  not  sound.    The  thesis  that  a  given  hemolytic 
immune  serum  contains  several  separate  specific  sensitizers  has  no 
experimental  justification.    The  conception  of  complement  devia- 
tion due  to  an  excess  of  sensitizer  is  purely  hypothetical. 

XL  The  short  duration  of  the  passive  immunity  afforded  by 
injecting  an  immune  serum  from  an  alien  species  would  seem  to  be 
due  to  the  fact  that  the  recipient  generally  elaborates  an  antago- 
nistic substance.  The  effect  of  this  substance  is  not  directed 
especially  against  the  antibody  that  gives  immunity,  but  in  a 


PROPERTIES  OF  ANTISENSITIZERS.  311 

general  way  against  any  and  all  sensitizers,  normal  or  specific,  present 
in  the  alien  serum.  The  production  of  this  antagonistic  substance 
is  not  in  any  way  dependent  on  the  identity  or  even  on  a  close 
relation  in  composition  between  the  cells  of  the  animal  body  and 
the  substances  injected.  There  is  no  reason,  in  other  words,  to 
assume  the  existence  of  common  receptors. 


XV.    RESEARCHES   ON  THE  AGGLUTINATION  OF  RED 
BLOOD  CELLS  BY  CHEMICAL  PRECIPITATES  AND 
ON  THE  SUSPENSION  OF  SUCH  PRECIPI- 
TATES IN  COLLOIDAL  MEDIA.* 

BY  DR.  OCTAVE  GENGOU. 

Colloidal  substances  play  so  important  a  role  in  vital  phenomena 
that  biologists  have  been  following  with  great  interest  the  advances 
made  by  chemistry  and  physics  in  our  knowledge  of  these  substances. 
Any  analysis  of  the  properties  of  organic  colloids  has  been  extremely 
complicated  by  our  ignorance  of  their  composition;  for  this  reason 
we  have  resorted  to  such  information  as  may  be  gained  from  a 
study  of  simpler  colloids.  We  have,  in  general,  drawn  our  con- 
clusions as  regards  organic  colloids  from  the  rules  that  have  been 
drawn  from  the  better-known  reactions  with  inorganic  colloids. 
One  of  the  best-known  phenomena  afforded  by  these  two  groups 
of  substances  is  agglutination.  Works  dealing  with  this  phe- 
nomenon are  numerous,  and  the  recent  publications  of  Perrin  on 
colloidal  substances  have  stimulated  biologists  to  a  most  active 
study  of  it. 

Although  the  agglutination  of  bacteria  and  of  red  blood  cells  by 
sera  has  been  investigated  with  much  diligence,  the  essential  prin- 
ciples of  the  reaction  are  still  a  mystery.  The  apparent  analogy 
between  agglutination  and  the  precipitation  of  colloids  has  given 
rise  to  the  hope  that  a  careful  analysis  of  this  latter  phenomenon 
may  lead  to  an  explanation  of  agglutination.  The  works  dealing 
either  with  the  precipitation  of  colloids  or  the  agglutination  of  red 
blood  cells  by  suspensions  (that  is,  by  colloids  or  chemical  precipi- 
tates) have  been  very  numerous  since  the  publications  of  Perrin. 

Landsteiner  and  Jagic  |  were,  we  believe,  the  first  to  draw  atten- 

*  Recherches  sur  I'agglutination  des  globules  rouges  par  les  precipites  chimiquea 
et  sur  la  suspension  de  ces  precipite"s  dans  les  milieux  colloidaux.  Annales  de 
1'Institut  Pasteur,  XVIII,  1904,  678. 

f  Landsteiner  and  Jagic,  Wien.  klin.  Wochenschr.,  1904,  No.  3. 

312 


AGGLUTINATION  OF  RED  BLOOD  CELLS.  313 

tion  to  the  fact  that  red  blood  cells  may  be  agglutinated  by  a  well- 
defined  colloid,  namely,  by  colloidal  silicic  acid.  Shortly  afterward 
we  ourselves*  reported  examples  of  agglutination  and  hemolysis 
of  red  blood  cells  by  means  of  such  chemical  precipitates  as  CaFl2 
and  BaS04.  Girard  Mangin  and  Henri  f  have  since  studied  this 
question  in  a  very  exhaustive  and  careful  manner  with  different 
colloids. 

As  we  have  already  demonstrated,  certain  chemical  precipitates 
as  well  as  colloids  agglutinate  red  blood  cells,  provided  the  latter 
are  washed  free  of  serum;  this  agglutination  is  followed  by  hemol- 
ysis, which  latter  phenomenon  we  have  referred  to  in  our  first  article. 
This  agglutination  and  hemolysis  is  inhibited  by  the  presence  of 
even  very  small  amounts  of  serum. 

In  the  present  article  we  shall  consider  simply  the  agglutination 
of  corpuscles  and  at  some  later  period  take  up  their  laking.  Mme. 
Girard-Mangin  and  V.  Henri  found  that  the  agglutination  of  red 
blood  cells  is  brought  about  by  negative  as  well  as  by  positive  col- 
loids. Red  blood  cells,  however,  have  a  negative  electric  charge,  as 
is  shown  by  the  fact  that  they  are  deposited  at  the  anode.  The 
fact  that  a  negative  emulsion  (corpuscles)  may  be  agglutinated  by 
equally  negative  colloids  evidently  does  not  coincide  with  the 
general  ideas  that  we  have  gained  concerning  the  action  of  colloids 
having  effect  on  one  another  or  other  colloids  with  the  same 
electric  charge.  A  mixture  of  colloids  having  the  same  electrical 
charge  indeed  causes  no  flocculation;  both  colloids  remain  in  sus- 
pension, $  and  so  Mme.  Girard-Mangin  and  V.  Henri  did  not 
conclude  that  the  agglutination  of  red  blood  cells  by  colloidal 
substances  is  due  to  a  direct  interaction  of  these  substances.  They 
believe  that  this  agglutination  is  only  an  indirect  and  secondary 
affair;  they  think  that  the  corpuscle  is  passive  in  the  phenomenon 
and  that  the  active  functions  are  exerted  by  the  colloidal  substances 
on  the  one  hand  and  by  endocorpuscular  salts  liberated  by  the  cor- 
puscles on  the  other. 

It  is  true,  indeed,  that  certain  colloidal  substances,  as,  for  example, 
those  studied  by  Mangin  and  Henri,  are  flocculated  by  electrolytes. 
When  red  blood  cells  are  left  in  normal  salt  solution  they  liberate 

*  Gengou,  Comptes  rend  us  de  1'Acad.  des  Sciences,  April  11,  1904. 

t  Mme.  Girard-Mangin  and  V.  Henri,  Soc.  de  Biol.,  1904,  Nos.  19,  20,  21, 24, 25. 

J  Henri,  Lalou,  Mayer  and  Stodel,  Soc.  de  Biol.,  1903,  Dec.  19. 


314  STUDIES  IN   IMMUNITY. 

a  certain  part  of  their  salts  and  it  is  perfectly  evident  that  these 
salts  in  the  course  of  their  diffusion  from  within  the  blood  cells  will 
be  more  abundant  in  the  pericorpuscular  zone  near  the  corpuscle 
than  in  the  fluid  between  the  corpuscles  at  a  relatively  considerable 
distance  from  the  corpuscles.  And  so  Mangin  and  Henri  think  that 
colloidal  substances  mixed  with  a  suspension  of  corpuscles  will 
by  preference  be  precipitated  in  the  more  concentrated  pericor- 
puscular zone. 

Following  this  first  stage,  a  second  would  occur,  during  which  the 
particles  of  colloidal  substances  that  have  been  flocculated  about 
the  corpuscles  by  means  of  the  endocorpuscular  salts  would  col- 
lect into  masses  and  drag  the  corpuscles  with  them;  hence  would 
result  the  formation  of  masses  composed  of  colloids  and  corpuscles. 

According  to  this  theory  the  agglutination  of  red  blood  cells 
by  colloidal  substances  depends  on  a  preliminary  precipitation  of 
these  colloids  about  the  corpuscles  by  means  of  the  diffused  electro- 
lytes from  within  the  corpuscles.  The  corpuscles  remain  quite 
passive  during  the  entire  phenomenon  and  are  simply  brought 
together  by  means  of  the  collection  of  particles  of  colloidal  sub- 
stances that  have  been  precipitated  about  them. 

We  do  not  believe  that  the  phenomenon  of  agglutination  of 
corpuscles  by  colloids  should  be  interpreted  in  this  way;  we  think 
rather  that  the  agglutination  is  due  to  a  direct  action  of  one  of 
these  elements  on  the  other.*  We  think,  indeed,  that  we  are  justi- 
fied in  applying  the  conclusions  drawn  from  the  facts  we  have 
observed  with  chemical  precipitates  to  the  agglutination  of  cor- 
puscles by  colloids.  According  to  the  opinion  of  Bredig  f  these 
two  substances  are  of  the  same  order  and  differ  from  one  another 
simply  in  a  variation  in  size  of  their  particles.  This  opinion  indeed 
is  shared  in  so  far  as  the  present  phenomena  are  concerned  by 
Landsteiner  and  Jagic  J  and  by  Mangin  and  Henri. § 

It  is  to  be  noted  in  the  first  place  that  the  colloids  studied  by  these 
latter  authors  are  very  susceptible  to  flocking  by  electrolytes; 

*  It  is  evident  that  there  is  no  dispute  about  the  well-known  action  of  salts  in 
the  phenomenon  of  agglutination  in  general;  we  are  dealing  here  simply  with  the 
function  attributed  by  Mangin  and  Henri  to  the  salts  that  have  diffused  from  the 
red  blood  cells  on  their  agglutination  by  colloids. 

t  Bredig,  Anorganische  Fermente. 

j  Landsteiner  et  Jagic,  Munch,  med.  Woch.,  No.  27,  1904. 

§  Mme.  Girard-Mangin  et  V.  Henri,  Soc  de  Biol.,  No.  19,  1904. 


AGGLUTINATION  OF  RED   BLOOD  CELLS.  315 

they  may,  therefore,  be  easily  flocculated  by  the  electrolytes  diffused 
from  the  corpuscles,  but  it  does  not  follow  that  this  explanation 
serves  to  explain  their  agglutinating  power  over  corpuscles. 

It  is  not  essential  for  a  substance  to  be  precipitated  by  salts 
coming  from  corpuscles  in  order  that  the  agglutination  by  this 
substance  be  possible.  We  have  already  noted  that  barium  sul- 
phate agglutinates  corpuscles  readily  and  barium  sulphate,  as  we 
know,  is  much  more  susceptible  to  gravity  than  it  is  to  electro- 
lytes. It  is  no  more  flocculated  by  a  strong  concentration  of  salt 
solution  than  it  is  by  distilled  water,  in  which  latter  solution  it 
sediments  rather  rapidly. 

We  have  undertaken  to  determine  whether  a  fine  suspension 
that  is  flocculable  by  electrolytes  can  subsequently  agglutinate 
corpuscles  after  having  been  flocculated. 

For  this  purpose  we  have  used  calcium  fluoride,  which  is  a  pre- 
cipitate of  rather  colloidal  appearance  flocculable  by  0.8  per  cent 
NaCl.  We  find  that  calcium  fluoride  that  has  been  previously 
flocculated  by  0.8  per  cent  or  by  2  per  cent  sodium  chloride  will 
still  agglutinate  corpuscles  in  0.6  per  cent  salt  solution  quite  as  well 
as  if  it  were  in  suspension  in  distilled  water.  If  the  saline  con- 
centration is  increased  to  3  per  cent  the  calcium  fluoride  so  treated 
no  longer  agglutinates  as  well.  This  would  prove  simply,  accord- 
ing to  our  opinion,  that  the  very  dense  accumulated  masses  of 
colloidal  precipitates  are  less  effective  on  corpuscles,  owing  to  the 
fact  that  with  such  masses  an  intimate  mixture  of  the  corpuscles 
and  the  calcium  fluoride  is  no  longer  possible.  When  the  masses, 
on  the  contrary,  are  not  so  dense  (for  example,  CaFl2  in  sodium 
chloride,  0.8  per  cent  or  2  per  cent)  and  consequently  more  easily 
broken  up,  the  effect  of  the  suspension  on  the  corpuscles  is  only 
slightly  attenuated.  We  should  not  conclude  from  these  facts, 
however,  that  the  agglutination  of  corpuscles  by  calcium  fluoride 
is  possible  only  when  the  state  of  dissociation  of  this  substance  is 
such  that  it  may  be  flocculated  by  the  salts  about  corpuscles  as  they 
pass  out,  but  simply  that  the  greater  the  intimacy  of  mixture 
between  the  corpuscles  and  the  calcium  fluoride,  the  more  intense 
the  agglutination.* 

*  It  is  probable  that  the  same  holds  true  for  the  colloidal  substances,  which 
become  distinctly  less  active,  as  they  are  less  easily  flocculated  by  electrolytes.  Mme. 
Girard-Mangin  and  V.  Henri  asserted  that  flocculated  colloids  no  longer  aggluti- 


316  STUDIES   IN   IMMUNITY. 

If  colloids  are  flocculated  about  corpuscles  before  agglutinating 
them,  as  Mme.  Girard-Mangin  and  V.  Henri  think,  such  a  concen- 
tration depends  on  the  difference  of  saline  concentration  between 
the  rich  pericorpuscular  zone  and  the  relatively  weak  fluid  between 
the  corpuscles.  If  this  difference  in  concentration  were  eliminated, 
there  would  be  no  reason  for  the  colloids  to  flocculate  about  the 
corpuscles,  and  consequently  the  agglutination  of  the  latter  by  the 
colloids  would  be  nil  or,  at  least,  very  much  diminished.  We  have  en- 
deavored to  determine  whether  such  a  result  may  be  verified  experi- 
mentally. We  introduce  in  a  large  volume  of  normal  saline  a  certain 
amount  of  well-washed  red  blood  corpuscles  and  allow  the  mixture 
to  stand  for  3  or  4  hours,  taking  care  to  shake  it  from  time  to 
time.  It  is  evident  that  the  longer  a  diffusion  of  intracorpuscular 
salts  goes  on  the  more  will  the  fluid  between  the  corpuscles  in- 
crease in  tonicity  and  the  more  perfect  will  the  equilibrium  between 
this  fluid  and  the  pericorpuscular  zone  become.  And,  what  is 
more,  in  proportion  as  the  corpuscles  lose  their  electrolytes,  the 
concentration  of  the  fluid  surrounding  them  becomes  more  nearly 
equal  to  their  tonicity.  Consequently,  after  a  certain  period  there 
will  be  an  equilibrium  between  the  corpuscles  and  the  surrounding 
fluid.  If  we  ccntrifugalize  at  this  point  and  decant  the  supernatant 
fluid,  we  shall  have  a  fluid  A  (salt  solution  plus  diffused  salts  from 
the  corpuscles)  and  the  red  blood  cells  D,  which  have  lost  their 
salts. 

Let  us  place  in  tube  1  a  given  amount  of  fluid  A  and  in  tube  2 
an  equal  amount  of  salt  solution;  we  then  add  to  both  the  same 
amount  of  CaFl2  and  allow  the  mixtures  to  stand  for  a  half  hour 
in  order  to  allow  the  intracorpuscular  salts  in  fluid  A  to  act  upon 
the  suspension.  We  then  add  to  each  tube  the  same  amount  of 
corpuscles  D.  Agglutination  of  the  corpuscles  immediately  takes 
place  and  there  is  no  difference  in  its  intensity  in  the  two  tubes, 
although  the  experimental  conditions  differ.  It  is  indeed  to  be 

nate  corpuscles.  Landsteiner  and  Jagic,  however,  find  that  the  previous  floccu- 
lation  of  colloids  does  not  diminish  their  agglutinating  property  for  corpuscles. 
It  is  possible  that  both  experimenters  are  correct,  and  that  one  may  flock  out 
colloids  at  various  stages  and  so  obtain  colloids  that  are  either  agglutinating  or 
not  so,  in  accordance  with  whether  the  electrolytes  have  formed  loose  or  dense 
clumps,  that  is  to  say,  depending  on  whether  a  mixture  of  the  colloids  with  the 
corpuscles  is  or  is  not  possible. 


AGGLUTINATION   OF  RED  BLOOD   CELLS.  317 

noted  that  in  this  experiment  we  used  D  corpuscles  and  not  fresh 
corpuscles.  The  reason  that  we  have  employed  D  corpuscles  is 
because  they  have  been  for  a  long  time  in  contact  with  fluid  A  and 
supposedly  are  very  similar  in  tonicity  to  this  fluid,  so  that  they 
have  nothing  more  to  give  out  into  it.  Under  these  conditions  we 
should  not  expect  the  pericorpuscular  zone  to  be  richer  in  salts 
than  is  the  fluid  between  the  corpuscles.  In  tube  2,  on  the  con- 
trary, D  corpuscles  are  placed  in  contact  with  saline  solution  that 
does  not  contain  the  corpuscular  salts,  and  their  tonicity  is  there- 
fore higher  than  that  of  the  salt  solution.  We  should  expect 
them,  therefore,  to  diffuse  their  own  salts  and  to  create  a  pericor- 
puscular zone  that  is  more  concentrated  than  the  fluid  between 
the  corpuscles.  To  sum  up,  in  tube  1  we  have  a  pericorpuscular 
zone  and  an  intercorpuscular  fluid  of  the  same  concentration;  in 
tube  2  we  have  a  pericorpuscular  zone  that  is  more  concentrated 
than  between  the  corpuscles.  The  agglutination  of  the  corpuscles 
by  calcium  fluoride  should  therefore  be  more  intense  in  tube  2 
than  in  tube  1  if  the  salts  in  the  pericorpuscular  zone  play  any 
role  in  the  phenomenon. 

And  there  would  be  still  another  reason  why  the  intensity  of  the 
agglutination  should  differ  in  the  tubes  in  our  experiment  if  this 
hypothesis  were  true.  As  we  introduced  CaFl2  some  time  before 
the  corpuscles,  this  suspension  was  subjected  to  the  flocculating 
effect  of  salt  solution  in  tube  2  and  to  the  effect  of  fluid  A  in  tube  1. 
As  fluid  A  contains  the  intracorpuscular  salts  it  should  have  more 
flocculating  action  than  salt  solution,  and  consequently  CaFl2, 
being  more  markedly  flocculated  in  tube  1,  should  in  tube  2  have 
less  effect  on  the  corpuscles.  As  we  have  already  seen,  the  results 
do  not  justify  this  conception,  and,  what  is  more,  CaFl2is  not  floc- 
culated in  tube  1  any  more  than  it  is  in  tube  2.  In  other  words,  the 
supposed  increase  of  salt  in  tube  1  from  the  corpuscles  does  not 
give  any  more  marked  flocculation  than  does  salt  solution. 

This  experiment  demonstrates  that  the  agglutination  of  cor- 
puscles by  CaFl2  does  not  lose  in  intensity  when  the  flocculating 
effect  that  the  pericorpuscular  zone  might  have  on  the  suspension 
is  reduced  to  the  minimum. 

The  objection  may  be  raised  that  the  diffusion  of  salts  from  the 
corpuscles  is  never  pushed  sufficiently  in  such  an  experiment  and 


318  STUDIES  IN   IMMUNITY. 

that  the  saline  concentration  of  the  pericorpuscular  zone  is  under 
any  condition  sufficient  to  bring  about  a  precipitate  of  the  colloid 
or  of  the  suspension  around  the  corpuscles.  We  have  noted  a  fact 
in  our  first  article  that  answers  this  objection:  we  caused  a  diffusion 
of  all  the  substances  that  the  corpuscles  contain.  For  this  pur- 
pose we  laked  corpuscles  with  distilled  water  and  then  washed  them 
several  times  in  0.7  per  cent  salt  solution.  As  a  result  of  several 
such  washings  we  presumed  that  the  saline  tonicity  of  the  strornata 
and  consequently  of  the  pericorpuscular  zone  should  not  exceed 
that  of  the  fluid  between  the  corpuscles,  and  that,  consequently, 
with  such  stromata  we  have  to  deal  simply  with  the  sodium  chlorid 
in  salt  solution.  Such  stromata  as  they  contain  no  intracorpuscular 
salts  should  not  be  agglutinable  by  a  colloid  or  by  a  chemical  sus- 
pension. We  find,  however,  that  on  adding  CaFl2  to  such  stromata 
an  agglutination  takes  place  which  is  quite  comparable  with  that 
of  red  blood  cells ;  the  CaFl2,  moreover,  may  be  replaced  by  barium 
sulphate  or  by  colloidal  ferric  hydroxide. 

We  see  no  reason  for  supposing  that  the  agglutination  of  stro- 
mata by  colloids  and  suspensions  is  to  be  explained  in  any  different 
way  than  that  of  red  blood  cells.  In  fact  the  agglutination  of 
stromata  by  such  a  suspension  as  CaFl2  follows  in  all  its  details  the 
phenomena  present  in  the  agglutination  of  red  blood  cells.  In 
both  instances  the  intensity  of  the  agglutination  depends  on  the 
amount  of  CaFl2  employed;  the  maximum  agglutination  occurring 
with  an  optimal  dose,  a  smaller  or  larger  amount  giving  rise  to  less 
agglutination.  In  each  instance,  moreover,  the  addition  of  a  small 
amount  of  serum  prevents  agglutination.  The  phenomena  are 
absolutely  interchangeable,  which  shows,  according  to  our  opinion, 
that  in  agglutination  of  corpuscles  by  a  suspension  the  salts  from 
the  corpuscles  do  not  first  flocculate  with  the  suspensions  in  order 
to  bring  about  agglutination.  The  fundamental  action  takes  place 
between  the  albuminoid  substances  (stromata)  of  the  corpuscles  and 
the  suspensions  in  question.* 

*  We  find,  indeed,  that  agglutination  of  stromata  may  be  produced  by  barium 
sulphate  whether  suspended  in  distilled  water  or  in  2  per  cent  salt  solution,  and 
it  seems  difficult  to  imagine  that  a  suspension  that  is  so  little  affected  by  elec- 
trolytes should  be  influenced  by  the  traces  of  salts  that  might  come  from  the 
stromata.  Mangin  and  Henri  have  noted  similar  phenomena  in  the  agglutination 


AGGLUTINATION  OF  RED   BLOOD  CELLS.  319 

There  is,  to  be  sure,  one  conclusion  in  the  theory  of  Mangin  and 
Henri  which  seems  experimentally  verified,  but  as  we  shall  see 
the  verification  is  only  apparent  and  their  conclusion  incorrect. 
This  is  the  subject  in  point.  If  it  is  true  that  the  salts  within 
corpuscles  have  something  to  do  with  flocculating  colloids  and  sus- 
pensions about  the  corpuscles,  on  extracting  the  salts  from  these 
corpuscles  by  laking,  we  should  obtain  a  fluid  L,  which,  when  the 
stromata  are  removed,  will  prevent  the  agglutination  of  fresh  cor- 
puscles by  a  suspended  precipitate.  This  in  particular  would  be 
true  if  the  suspension  were  placed  in  fluid  L  before  the  fresh  cor- 
puscles were,  thus  allowing  it  to  be  flocculated  by  the  corpuscular 
salts  present  in  the  fluid.  We  proceed,  then,  by  laking  blood  cor- 
puscles with  distilled  water  and  subsequently  adding  enough  sodium 
chlorid  to  restore  its  tonicity  to  that  of  physiological  salt  solution, 
and  we  then  separate  the  stromata  by  centrifugalization  and 
decant  the  supernatant  fluid,  which  contains  all  the  substances 
that  the  corpuscles  liberated.  To  a  given  amount  of  this  fluid  we 
add  a  known  quantity  of  CaFl2,  of  barium  sulphate  or  of  colloidal 
ferric  hydroxide  and,  after  a  short  period,  we  add  to  each  tube  a 
small  amount  of  fresh  washed  corpuscles.  No  agglutination  occurs. 
The  laked  fluid,  then,  deprived  of  stromata  completely  prevents 
the  agglutination  of  other  corpuscles  either  by  suspensions  or  by 
ferric  hydrate. 

At  first  sight  this  fact  agrees  very  well  with  Mangin  and  Henri's 
ideas,  but  it  is  not,  however,  explicable  as  we  see  it  by  their  theory. 
If  the  inhibiting  effect  of  this  fluid  is  in  reality  due  to  diffused 
intracorpuscular  salts,  it  must  be  due  to  the  precipitation  of  the 
colloids  or  suspensions  by  these  salts,  which  precipitation  in  this 
case  would  occur  without  the  subsequent  addition  of  corpuscles. 
We  find,  however,  no  precipitation  of  colloids  or  suspensions  by 
this  laked  fluid;  the  reverse  indeed  is  found.  Salt  solution  rapidly 
flocculates  CaFl2  and  ferric  hydrate  and  allows  barium  sulphate  to 
sediment  rather  quickly,  but  fluid  L,  deprived  of  stromata,  keeps 
these  substances  in  suspension;  even  the  ferric  hydrate,  which  is 
so  susceptible  to  the  presence  of  salts.  Since  the  electrolytes  in 

of  corpuscles  by  colloids;  they  find  that  stromata  are  also  agglutinable  by  these 
substances.  Under  such  conditions  we  see  no  reason  for  attributing  an  active 
function  to  the  corpuscular  salts,  as  do  these  authors. 


320  STUDIES  IN  IMMUNITY. 

this  fluid  can  only  have  a  flocculating  action  they  are  evidently 
not  responsible  for  producing  this  suspension.* 

We  believe  (although  our  opinion  is  based  on  no  irrefutable 
experimental  proof)  that  if  ferric  hydrate  and  CaFl2  fail  to  be  pre- 
cipitated by  the  electrolytes  of  fluid  L,  and  if  barium  sulphate 
remains  in  a  state  of  fine  suspension  in  this  fluid,  it  is  due  to  the 
fact  that  they  are  held  in  suspension  by  the  colloidal  substances  of 
an  albuminous  nature  that  have  come  from  the  corpuscles  through 
laking.  The  phenomenon,  indeed,  is  identical  with  the  dissociat- 
ing effect  of  serum  on  barium  sulphate,  to  which  we  have  already 
referred  and  which  we  shall  presently  again  consider.  This  dis- 
sociating power  in  the  laked  fluid  can  be  exhausted,  as  can  the 
same  power  in  serum,  and  if  we  presume  that  the  precipitate  is  due 
to  some  substance  we  may  suppose  that  this  substance  can  be 
removed  from  the  laking  fluid.  As  a  proof  let  us  add  a  rather  large 
amount  of  CaFl2  to  fluid  L  containing  no  stromata;  after  15  minutes' 
contact  we  will  remove  the  CaFl2  by  centrifugalization  and  obtain, 
on  decanting,  a  fluid  A  which  differs  from  fluid  L  only  in  having 
been  subjected  to  a  large  amount  of  fluoride.  We  find  that  the 
sedimentation  and  flocculation  of  the  various  substances  to  which 
we  have  referred,  although  prevented  by  fluid  L,  occur  very  readily 
in  fluid  A.  And,  what  is  more,  an  agglutination  of  red  blood  cells 
by  suspensions  and  by  ferric  hydrate  takes  place  perfectly  in  A, 
although  it  does  not  occur  in  L. 

The  large  amount  of  CaFl2,  then,  has  removed  something  from 
fluid  L  that  prevents  the  flocculation  of  CaFl2  and  of  ferric  hydrate 
by  electrolytes  as  well  as  the  sedimentation  of  barium  sulphate. 
The  property  of  preventing  the  agglutinating  of  corpuscles  by 
means  of  these  three  substances  has  also  been  removed  from  fluid  L 
by  this  treatment.  We  believe  that  thie  substance  or  substances 
are  albuminous  colloids  that  come  from  the  corpuscles  as  a  result 
of  the  laking,  and  we  base  this  hypothesis  on  the  results  obtained 

*  Neisser  and  Friedemann*  have  noted,  it  is  true,  that  colloids  flocculated  by 
a  given  dose  of  salts  no  longer  give  such  a  result  if  the  amount  of  salt  is  increased. 
In  their  experiments,  however,  they  deal  with  the  salts  of  heavy  metals  and  they 
have  compared  this  fact  to  the  formation  of  colloidal  hydrate  by  hydrolysis  of 
its  electrolytes.     This  evidently  is  not  the  case  with  the  salts  obtained  from  red 
blood  cells. 

•  Neisser  and  Friedemann,  Munch,  med.  Woch.,  1904,  No.  11. 


AGGLUTINATION  OF  RED  BLOOD  CELLS.  321 

by  studying  the  inhibiting  effect  of  serum  on  the  agglutination  of 
corpuscles  by  suspensions.* 

Once  these  colloids  are  removed  we  have  a  fluid  (A)  that  allows 
the  flocculation  of  CaFl2  and  of  ferric  hydrate  by  the  intracorpus- 
cular  salts  present  in  this  laked  fluid.  This  flocculation,  as  we  know, 
takes  place.  This  fact,  however,  does  not  prevent  it  from  agglu- 
tinating subsequently  added  corpuscles.  These  experiments  show, 
as  we  believe,  that  the  intracorpuscular  salts  extracted  by  laking 
do  not  antagonize  the  agglutination  of  fresh  corpuscles  by  sus- 
pensions or  colloids;  we  have  just  recently  seen,  moreover,  that 
the  stromata,  deprived  of  intracorpuscular  salts,  are  agglutinable 
by  colloids  and  suspensions  as  well  as  are  corpuscles. 

As  a  result  of  this  we  conclude  that  in  order  to  explain  the  agglu- 
tinating effect  of  these  suspensions  with  which  we  have  been  deal- 
ing, on  corpuscles,  it  is  not  necessary  to  suppose  that  a  flocculation 
of  them  by  means  of  the  intracorpuscular  salts  takes  place.  We 
have  indeed  seen,  first,  that  this  agglutination  is  likewise  possible 
by  suspensions  that  have  previously  been  flocculated  by  electrolytes ; 
and  second,  that  there  is  no  change  when  the  excess  of  salts  in  the 
pericorpuscular  zone  of  the  corpuscles  is  reduced  to  the  minimum 
so  as  to  annihilate  the  hypothetical  flocculating  effect  of  this  zone 
on  the  suspension;  and  third,  the  phenomenon  of  the  agglutination 
of  stromata  is  identical  with  that  of  corpuscles;  fourth,  the  sen- 
sitivity of  these  suspensions  to  the  flocculating  action  of  salts  is 
not  a  necessary  preliminary  to  their  agglutinating  property  toward 
corpuscles.  Inasmuch  as  all  the  phenomena  of  the  agglutination 
of  corpuscles  by  a  suspension  take  place  just  as  well  with  colloids, 
we  think,  whether  or  not  these  latter  are  flocculated  by  diffused 
intracorpuscular  salts,  that  this  has  no  effect  on  their  agglutinat- 
ing property  over  red  blood  cells.  We  have  endeavored  to  prove 
this  fact  more  directly.  We  have  attempted  to  produce  phenom- 
ena of  agglutination  analogous  to  those  already  described  by  allow- 
ing our  suspensions  to  act  upon  an  emulsion,  the  particles  of  which 
contain  no  salts  that  might  be  diffused,  in  place  of  red  blood  cells. 
For  this  purpose  we  have  used  emulsions  of  oil  prepared  by 

*  We  have  already  noted  in  our  first  article  that  although  serum  prevents  the 
agglutination  of  suspensions  with  corpuscles  this  inhibiting  property  may  be 
removed  by  treating  it  previously  with  a  large  dose  of  CaFl2  or  of  barium  sulphate. 


322  STUDIES  IN  IMMUNITY. 

adding  5  drops  of  olive  oil  to  10  c.c.  of  distilled  water  plus 
0.001  of  a  cubic  centimeter  of  sodium  carbonate.  Such  feebly 
alkaline  emulsions  may  be  neutralized  or  even  acidified  without 
showing  any  change.  In  our  experiments  we  have  used  such 
emulsions  neutralized  as  carefully  as  possible.  Working  with  them 
we  find  that  the  tiny  drops  of  oil  agglutinate  on  the  addition  of 
suspensions  of  CaFl2  and  barium  sulphate  and  also  of  colloidal 
ferric  hydrate.  The  flocks  obtained  in  this  manner  are  found 
microscopically  to  consist  of  a  suspension  of  the  hydrate  with  drops 
of  oil  scattered  among  them.  It  is  found,  moreover,  that  the  addi- 
tion of  a  small  amount  of  serum  prevents  the  agglutination  of 
oil  corpuscles  by  suspensions  and  by  ferric  hydrate  in  the  same 
way  as  it  does  the  agglutination  of  red  blood  cells  by  chemical 
precipitates. 

Inasmuch  as  these  two  forms  of  agglutination  apparently  agree, 
we  believe  that  their  respective  causes  are  due  to  the  same  factors 
and  that  the  agglutination  of  red  blood  cells  by  suspensions  and 
colloids  is  not  an  indirect  result  of  the  effect  of  electrolytes  on 
these  suspensions,  but  due  to  a  direct  effect  of  one  element  on  the 
other. 

We  have  just  seen  that  certain  suspensions  agglutinate  blood 
cells.  If  in  place  of  the  corpuscles  we  add  serum  to  either  of  these 
two  suspensions  we  find  that  CaFl2  becomes  clumped,  while  barium 
sulphate  is  unaffected.  Suspended  in  salt  solution  barium  sulphate 
sediments  rapidly  to  the  bottom  of  the  tube.  In  serum,  on  the  con- 
trary, it  does  not  fall  or  falls  only  very  slowly,  and  remains  in  the 
fluid  in  the  form  of  a  fine  suspension.  This  dissociating  effect  of 
serum  on  barium  sulphate  is  very  marked  and  evident  even  on 
the  addition  of  1  drop  of  serum  diluted  1-20  to  1  c.c.  of  0.8  per 
cent  salt  solution  containing  4  drops  of  an  emulsion  of  barium 
sulphate. 

We  have  then,  on  the  one  hand,  an  agglutination  (barium  sul- 
phate plus  corpuscles)  and  on  the  other  hand  quite  a  different 
phenomenon,  namely,  a  dissociation  of  the  suspension  (barium 
sulphate  plus  serum).  It  is  generally  supposed  that  the  albuminous 
substances  of  serum  are  in  a  state  of  colloidal  solution.  An  emul- 
sion of  corpuscles,  then,  is  the  same  as  serum  in  that  both  are  emul- 


AGGLUTINATION   OF  RED   BLOOD   CELLS.  323 

sions  of  albuminous  substances,  but  in  the  case  of  the  corpuscles 
the  particles  are  larger  than  in  the  case  of  serum.  Both  serum  and 
corpuscles  have  the  same  negative  electric  charge.* 

How  is  it,  then,  that  the  addition  of  a  given  substance — barium 
sulphate  —  produces  such  divergent  effects  in  these  two  similar 
emulsions?  Agglutination  and  dissociation  would  indeed  seem 
to  be  the  direct  opposites  of  one  another,  but  are  they  indeed  as 
different  as  at  first  seems?  Are  they  not  rather  two  different  results 
of  a  single  fundamental  phenomenon  appearing  either  in  the  form 
of  an  agglutination  or  of  a  dissociation,  as  the  case  may  be? 

The  agglutination  of  corpuscles  with  barium  sulphate  leads  us 
to  think  of  some  adhesion  between  the  suspension  and  the  cor- 
puscles. In  the  dissociation  of  barium  sulphate  by  serum,  is  there 
not  also  an  adhesion  between  the  suspension  and  the  albuminous 
particles  of  the  serum?  According  to  this  explanation  the  funda- 
mental phenomenon  would  be  adhesion  between  the  suspension  and 
the  corpuscles  or  the  albuminous  substances  of  serum;  this  adhe- 
sion, however,  would  be  followed  in  the  one  case  by  agglutination 
and  in  the  other  by  dissociation. 

The  following  experiments  seem  to  prove  the  accuracy  of  this 
hypothesis : 

EXPERIMENT  1.  If  there  is  some  adhesion  between  the  albuminous  particles  of 
serum  and  barium  sulphate,  the  dissociating  property  of  the  serum  for  the  powder 
should  have  a  definite  limit.  In  order  to  demonstrate  this  we  add  a  large  amount 
of  barium  sulphate  to  a  small  amount  of  serum.  We  begin  by  centrifugalizing  five 
tubes,  each  of  which  contains  2  c.c.  of  our  emulsion  of  sulphate,  and  then  throw  off  the 
supernatant  fluids.  One  of  these  sediments  is  then  suspended  in  the  serum  employed, 
namely  0.5  of  a  cubic  centimeter  of  fresh  horse  serum  diluted  with  1.5  c.c.  of  salt 
solution.  After  allowing  short  contact  the  tube  is  then  centrifugalized  and  the  second 
original  sediment  of  sulphate  is  suspended  in  the  supernatant  fluid  of  the  first  tube; 
this  procedure  is  repeated  with  the  third  and  fourth  sediment  and  so  on.  We  then 
add  to  the  serum  that  has  been  treated  in  this  manner  a  small  amount  of  barium  sul- 
phate and  find  that  this  serum  has  little  or  no  dissociating  property  for  the  suspension. 
Nor  does  it  agglutinate  CaFl2  or  inhibit  the  agglutination  of  corpuscles  by  either 
CaFl2  or  by  barium  sulphate.  The  colloidal  solution,  which  constitutes  the  serum, 
loses  its  albuminous  substance,  then,  as  a  result  of  contact  with  larger  amounts  of 
barium  sulphate,  just  as  an  emulsion  of  red  blood  cells  in  agglutinating  the  sulphate 
solution  loses  its  corpuscles. 

*  It  is  to  be  noted  that  although  Mangin  and  Henri  consider  serum  as  a  nega- 
tive colloid,  other  writers,  notably  Neisser  and  Friedemann,  are  doubtful  of  this 
fact  on  account  of  Hardy's  work. 

Mme.  Girard-Mangin  et  V.  Henri.,  Soc.  de  Biol.,  1904,  No.  24. 

Neisser  et  Friedemann,  1.  c. 


324  STUDIES   IN   IMMUNITY. 

EXPERIMENT  2  //  the  comparison  is  exact  it  must  be  true  that  dissociation  of 
barium  sulphate  by  serum  is  due  to  an  adhesion  of  the  colloidal  substances  of  the 
serum  similar  to  that  which  takes  place  between  the  corpuscles  and  sulphate  in  their 
agglutination.  Repeated  washings  in  salt  solution  of  these  masses  formed  by  the 
adhesion  of  corpuscles  and  suspension  lead  to  no  change  in  the  clumps.  Shall  we 
find  in  a  similar  way  that  barium  sulphate  that  has  been  dissociated  by  serum  will 
remain  dissociated  after  washing,  in  other  words,  that  the  adhesion  of  the  suspension 
to  the  dissociating  colloids  of  the  serum  will  persist?  Twelve  drops  of  our  emulsion 
of  barium  sulphate  are  mixed  with  a  large  dose  of  horse  serum,  that  is,  with  3  c.c.  of 
fresh  serum  diluted  1-4,  and  after  5  minutes  the  mixture  is  centnfugalized.  The 
sediment  is  subsequently  washed  in  salt  solution  until  the  wash  water  contains  no 
trace  of  free  serum,  as  evidenced  by  the  fact  that  on  boiling  it  does  not  become  white  and 
that  it  fails  to  dissociate  fresh  sulphate.  We  then  suspend  this  sulphate  treated  by 
serum  in  salt  solution  and  find  that  it  remains  in  the  form  of  an  emulsion.  This 
emulsion  differs  absolutely  from  fresh  barium  sulphate  in  salt  solution;  it  is  very 
colloidal  in  appearance  and  resembles  milk;  it  finally  clarifies,  but  without  any  evidence 
of  an  agglutination  of  particles,  since  a  slight  jar  suffices  to  restore  it  to  the  form  of 
an  emulsion. 

This  experiment  is  the  complement  of  the  preceding;  serum 
owes  its  dissociating  property  to  colloidal  substances  that  adhere 
to  the  barium  sulphate,  just  as  red  blood  cells  adhere  to  it. 

EXPERIMENT  3.  We  have  endeavored  to  demonstrate  more  directly  the  union 
that  occurs  between  the  colloids  of  serum  and  the  dissociated  suspensions.  As  we 
shall  see,  we  have  succeeded  in  liberating  or  separating  these  colloids  from  the  suspen- 
sion to  which  they  were  attached.  In  order  to  do  this  it  is  necessary  to  eliminate  the 
original  suspension  by  dissolving  it.  For  this  purpose  we  have  employed  tricalcium 
phosphate  as  an  emulsion  in  distilled  water  instead  of  barium  sulphate,  which  is 
difficult  to  dissolve.  This  tricalcium  phosphate  is  similar  to  barium  sulphate  in  that 
it  sediments  rather  rapidly  in  salt  solution  and  distilled  water,  but  remains  for  a  long 
time  as  an  emulsion  in  the  presence  of  horse  serum. 

Three  drops  of  our  emulsion,  for  example,  remain  dissociated  for  a  long  time  in 
1  c.c.  of  serum  diluted  1-10.  We  saturate  a  rather  large  dose  of  tricalcium  phosphate, 
for  example,  20  drops  of  our  emulsion,  with  4  c.c.  of  serum  diluted  1-4  in  salt  solution, 
and  after  15  minutes'  contact  centrifugalize  the  precipitate  and  wash  repeatedly  with  salt 
solution  until  the  wash  water  contains  no  free  serum.  TJie  phosphate  is  then  suspended 
in  as  much  salt  solution  as  the  amount  of  serum  employed  in  the  first  place  and  gives 
a  homogeneous  emulsion  impregnated  with  serum  as  in  the  case  of  barium  sulphate. 

We  place  1  c.c.  of  this  emulsion  in  tube  1,  and  an  equal  amount  of  fresh  phosphate 
in  tube  2,  a  corresponding  amount  of  fresh  phosphate  plus  1  c.c.  of  salt  solution  and 
a  very  small  amount  (0.025  c.c.)  of  serum  in  1  c.c.  of  salt  solution  in  tube  3.  We 
then  add  to  each  tube  one  drop  of  acetic  acid.  There  is  no  change  in  tube  3;  a  certain 
amount  of  the  ptiosphate  in  tubes  1  and  2  is  dissolved  and  the  rest  remains  suspended. 
Fifteen  minutes  later  these  two  tubes  are  centrifugalized  and  the  supernatant  fluid 
decanted.  These  fluids  differ  in  this  respect:  The  one  is  obtained  by  dissolving  the 
fresh  phosphate  (tube  2),  and  the  other  by  dissolving  phosphate  that  has  previously 
been  impregnated  with  the  dissociating  substance  of  serum  (tube  1).  //  we  have 
succeeded  in  liberating  the  dissociating  substances  in  tube  1  by  dissolving  the  phosphate 


AGGLUTINATION  OF  RED   BLOOD  CELLS.  325 

to  which  they  were  faced,  we  should  be  able  to  demonstrate  it  by  a  dissociation  of  barium 
sulphate  added  to  this  fluid.  This  indeed  proves  to  be  true:  barium  sulphate  added 
to  these  two  fluids  and  also  to  tube  3  is  found  to  sediment  rapidly  in  tube  2  and  to 
remain  dissociated  in  tube  1  and  in  tube  3.  Control  tubes  show  that  neither  the  wash 
water  nor  the  acetic  acid  have  the  slightest  dissociating  effect  on  barium  sulphate.  On 
choosing,  therefore,  a  suspension  that  is  dissociated  by  serum  and  easy  to  dissolve,  the 
combination  between  the  suspension  and  the  colloidal  substances  that  dissociated  it 
may  be  broken  up.* 

These  experiments  show,  it  seems  to  us,  that  the  phenomena  of 
agglutination  and  dissociation,  at  least  in  the  instances  we  have 
studied,  although  of  different  appearance,  are  nevertheless  due  to 
the  same  fundamental  fact,  namely,  the  affinity  of  the  suspension 
for  the  corpuscles  or  the  colloids  of  serum.  Why  are  so  widely 
different  phenomena  produced  under  such  apparently  closely 
related  conditions?  We  cannot  attempt  to  give  the  ultimate  reason 
for  this  difference,  but  will  simply  offer  an  hypothesis  that  we  have 
supported  by  a  few  facts.  Since  we  know  that  an  adhesion  occurs 
in  both  instances  between  a  suspension  and  the  particles  either 
of  the  corpuscle  suspension  or  of  the  serum,  the  question  arises  if, 
when  these  adhesions  are  produced,  the  respective  properties  of 
the  substances  employed  do  not  have  some  effect  on  the  result  of 
the  reaction?  As  we  know,  barium  sulphate  will  sediment  by  its 
own  weight.  If  we  add  corpuscles  which  are  particles  that  also 
tend  to  sediment,  to  barium  sulphate,  may  we  not  consider  the 
fact  that  the  combination  falls  to  the  bottom  of  the  tube  as  due  to 
the  tendency  of  barium  sulphate  to  sediment,  which  tendency  is 
only  slightly  modified  by  a  contrary  tendency  on  the  part  of  the 
corpuscles?  In  a  mixture  of  barium  sulphate  and  serum,  however, 
the  sulphate  meets  with  colloids  which  are  very  difficult  to  pre- 
cipitate and  the  tendency  of  barium  sulphate  to  sediment  would 
be  counterbalanced  by  a  marked  opposing  tendency.  If  this 
opposing  tendency  prevails,  a  combination  with  barium  sulphate 
would  not  sediment  at  all,  but  remain  dissociated  in  the  fluid,  as 
indeed  happens.  According  to  this  hypothesis  the  dissociation  of 
barium  sulphate  by  serum  would  be  due  to  the  result  of  a  struggle 

*  It  is  to  be  noted  that  if  hydrochloric  acid  is  used  in  place  of  acetic  acid  and 
the  phosphates  thus  totally  dissolved,  barium  sulphate  is  not  dissociated  by  the 
resultant  fluid;  it  is  also  to  be  noted  that  barium  sulphate  sediments  in  serum  to 
which  the  same  amount  of  hydrochloric  acid  has  been  added.  In  other  words, 
hydrochloric  acid  inhibits  the  dissociating  effect  of  the  serum. 


326  STUDIES  IN   IMMUNITY. 

between  opposing  influences.  And  if  this  is  true  we  should  be  able 
to  modify  the  outcome  of  the  struggle  by  enfeebling  one  or  the 
other  of  the  influences  present ;  and  this  we  have  done  in  the  follow- 
ing manner:  It  is  well  known  that  heating  serum  to  60  to  65  degrees 
brings  it  through  a  series  of  transitory  stages  to  a  dense  coagulum. 
This  coagulum  may  be  avoided  by  a  preliminary  dilution  of  the 
serum,  under  which  conditions  the  serum  is  still  coagulable  more 
or  less  completely,  according  to  the  degree  of  dilution,  but  never 
in  a  single  mass.  For  example,  if  we  leave  horse  serum,  that  has 
been  diluted  with  three  times  its  volume  of  salt  solution,  for  a  quarter 
of  an  hour  in  boiling  water,  we  obtain  a  whitish  fluid  containing 
albuminoids  coagulated  to  a  greater  or  less  extent,  but  not  in 
a  single  mass;  such  a  fluid  is  a  true  colloidal  solution,  but  in 
comparison  with  unheated  serum  it  represents  a  coagulated  solu- 
tion, the  particles  of  which  have  a  greater  tendency  to  clump  and 
sediment. 

We  now  prepare  two  tubes  which  contain  the  same  quantity  of 
barium  sulphate  in  the  same  volume  of  salt  solution  and  add  to 
one  a  given  dose  of  non-heated  serum  diluted  1-4  in  salt  solution, 
and  to  the  other  the  same  amount  of  the  same  diluted  serum  that 
has  been  heated  for  15  minutes  in  boiling  water.  The  barium 
sulphate  becomes  dissociated  in  the  first  tube,  but  in  the  second 
forms  clumps  that  are  quite  different  from  the  spontaneous  sedimen- 
tation in  salt  solution.  After  this  agglutination  is  finished  and 
the  clumps  have  fallen  to  the  bottom  of  the  tube,  the  supernatant 
fluid  has  lost  much  of  its  milky  appearance  and  the  subsequent 
addition  of  a  fresh  amount  of  barium  sulphate  leads  to  only  a  slightly 
increased  clarification  in  the  fluid,  the  agglutinating  property  of 
which  has  become  very  much  diminished.  The  agglutination  of 
barium  sulphate  by  heated  serum  is  due,  then,  to  a  combination 
between  the  suspension  and  the  colloidal  substances  which  give 
the  serum  its  milky  appearance.  Since  this  is  true  the  aggluti- 
nating property  may  be  removed  from  heated  serum  by  adding 
sufficiently  large  amounts  of  barium  sulphate.  We  collect  at  the 
bottom  of  the  tube  by  centrifugalization  the  sulphate  contained 
in  4  c.c.  of  our  emulsion  and  remove  the  supernatant  fluid  and  then 
suspend  the  sediment  in  a  mixture  containing  0.6  of  a  cubic  centi- 
meter of  horse  serum  plus  1.8  c.c.  of  salt  solution  (the  mixture 


AGGLUTINATION  OF  RED   BLOOD  CELLS.  327 

having  been  heated  for  15  minutes  in  boiling  water).  After  a  few 
minutes  we  again  centrifugalize. 

The  supernatant  fluid  is  absolutely  limpid  and  has  no  effect  on 
fresh  barium  sulphate  and  does  not  turn  white  on  boiling. 

To  sum  up,  we  have  seen  that  the  dissociation  of  barium  sul- 
phate by  serum  and  its  agglutination  by  red  blood  corpuscles  is 
clue  fundamentally  to  the  same  phenomenon,  namely,  the  adhesion 
of  the  sulphate  with  the  particles  of  the  serum  or  of  the  corpuscles, 
as  the  case  may  be.  We  have  offered  the  hypothesis  that  the 
result  of  such  a  combination  depends  on  the  tendency  of  the  par- 
ticles united  with  barium  sulphate  to  remain  in  suspension ,  and  we 
have  shown  that  by  diminishing  this  tendency  the  property  of 
the  sulphate  to  sediment  becomes  preponderant  and  leads  to  an 
agglutination  instead  of  a  dissociation.  This  hypothesis  is  sup- 
ported by  the  experiment  to  which  reference  has  just  been  made.* 

*  Two  objections  may  be  raised  which  it  may  be  well  to  meet  before  going 
farther.  It  might  be  possible  that  the  substances  causing  agglutination  or  dis- 
sociation with  barium  sulphate,  in  accordance  with  whether  the  serum  has  or  has 
not  been  heated,  are  not  the  same;  and  it  might  be  supposed  that  heat  has  simply 
caused  the  agglutinating  property  to  appear  by  destroying  the  dissociating  prop- 
erty. Such,  however,  does  not  seem  to  be  the  case.  We  find  that  on  treating 
a  certain  amount  of  fresh  serum  (0.5c.c.  plus  1.5  c.c.  of  salt  solution)  with  large 
amounts  of  barium  sulphate,  we  remove,  as  we  have  already  mentioned,  all 
the  dissociating  properties  of  the  serum.  If  the  diluted  serum  treated  in  this 
manner  is  then  heated  for  one-quarter  hour  to  100  degrees  with  a  tube  of  serum 
that  has  not  been  treated  with  barium  sulphate,  the  latter  becomes  milky  through 
coagulation  of  its  albuminous  substances,  whereas  the  first  tube  shows  no  appre- 
ciable whitening.  The  addition  of  this  latter  treated  serum  to  a  small  amount 
of  barium  sulphate  produces  little  or  no  agglutination.  By  removing  the  disso- 
ciating substance  from  fresh  serum  by  adding  large  doses  of  sulphate,  we  thus 
remove  at  the  same  time  its  property  of  agglutinating  the  sulphate  after  heating. 
This  renders  it  very  probable  that  the  different  actions  on  barium  sulphate  are 
due  to  the  same  substances. 

The  objection  may  also  be  raised  that  the  dissociating  property  of  serum  is 
retained  even  after  heating,  but  is  masked  by  the  agglutinating  properties  which 
this  heating  develops.  This,  however,  does  not  seem  to  us  to  be  correct.  We 
have  just  seen  that  the  agglutinating  properties  may  be  removed  from  heated 
serum  by  treating  it  with  large  doses  of  barium  sulphate,  and  it  may  also  be  noted 
from  the  doses  that  we  have  given  that  this  removal  is  much  easier  than  the 
removal  of  the  dissociating  property  from  unheated  serum.  Thus  we  find  that 
whereas  4  c.c.  of  our  emulsion  of  sulphate  suffices  to  remove  the  agglutinating 
property  from  2.4  c.c.  of  serum  diluted  1-4  and  heated  to  100  degrees,  10  c.c. 
of  the  same  emulsion  suffices  only  to  weaken  the  dissociating  properties  of  2  c.c. 
of  the  same  serum  diluted  1-4  and  unheated.  If  the  agglutinating  and  dissocia- 


328  STUDIES  IN  IMMUNITY. 

If  it  be  true  that  the  agglutination  of  barium  sulphate  and  heated 
serum  depends  on  the  diminution  of  the  colloidal  condition  of  the 
latter,  we  should  expect  to  obtain  different  phenomena,  in  accord- 
ance with  whether  we  add  a  large  or  small  amount  of  heated  serum 
to  a  given  amount  of  suspension.  We  put  the  same  amount  of 
barium  sulphate  (4  drops)  in  two  tubes  and  add  to  one  0.95  c.c.  of 
salt  solution  plus  0.05  c.c.  of  heated  serum  and  to  the  other  0.6  c.c. 
of  salt  solution  plus  0.4  c.c.  of  the  same  serum.  In  the  first  tube, 
containing  a  small  amount  of  serum,  a  fine  clumping  occurs,  but  the 
sulphate  remains  dissociated  in  the  second  tube.  The  loss  of 
dissociating  property  of  the  serum  by  heat  may  be  made  up  for, 
then,  by  adding  a  larger  amount  of  the  heated  serum. 

Not  only  will  a  large  dose  of  heated  serum  dissociate  fresh  barium 
sulphate,  but  it  will  also  restore  to  a  fine  suspension  sulphate  that 
has  been  previously  agglutinated  by  a  small  dose  of  serum.  We 
prepare  two  tubes  containing  each  0.95  of  a  cubic  centimeter  of 
salt  solution  and  0.05  c.c.  of  heated  serum  and  4  drops  of  our 
emulsion  of  sulphate-;  when  agglutination  has  become  complete,  to 
one  of  the  tubes  we  add  0.35  of  a  cubic  centimeter  of  the  same 
heated  serum  and  to  the  other  the  same  amount  of  salt  solution, 
and  shake.  The  agglutination  persists  in  the  second  tube,  but  the 
suspension  becomes  dissociated  in  the  first  tube;  the  first  tube  then 
looks  exactly  as  if  0.4  of  a  cubic  centimeter  of  heated  serum  had 
been  added  in  a  single  dose  to  4  drops  of  sulphate.* 

A  large  dose  of  heated  diluted  serum  is  necessary  to  dissociate 
barium  sulphate.  The  serum,  however,  may  be  heated  to  100 
degrees  in  such  a  way  as  to  preserve  its  dissociating  property  almost 

ting  properties  of  serum  were  due,  then,  to  different  substances,  we  should  be  able 
to  remove  the  first  by  a  small  dose  of  barium  sulphate  and  thus  leave  the  second 
intact.  We  may  then  treat  a  given  amount  of  heated  serum  with  an  amount  of 
sulphate  which  is  just  sufficient  to  remove  its  cloudiness;  in  this  way  we  remove  all 
the  agglutinating  properties,  but  we  find  that  no  trace  of  dissociating  property 
reappears. 

We  conclude,  then,  that  the  two  different  actions  of  serum  on  barium  sulphate 
are  due  to  the  same  substance  which  becomes  changed  by  heat. 

*  There  is  an  evident  resemblance  between  this  fact  and  the  observation  of 
Henri  and  Mayer,  who  found  that  the  clumps  resulting  from  an  agglutination  of 
two  colloids  of  different  charges  become  dissolved  in  an  excess  of  one  or  the  other 
of  these  colloids. 

*  Henri  and  Mayer.  Soc.  de  Biol.,  1904,  No.  19. 


AGGLUTINATION  OF  RED  BLOOD  CELLS.  329 

intact;  if  we  dilute  the  serum,  not  with  3  volumes  of  salt  solution, 
but  with  3  volumes  of  distilled  water  and  heat  for  15  minutes  to 
100  degrees,  the  dissociating  property  of  the  serum  for  suspensions 
is  almost  intact.  It  should  be  noted,  however,  that  whereas  serum 
diluted  in  salt  solution  and  heated,  becomes  white,  serum  diluted 
in  distilled  water  scarcely  changes  in  color,  that  is  to  say,  the  al- 
buminous substances  of  the  serum  are  very  incompletely  coagulated. 
This  modification  of  the  albuminous  substances  is  indispensable, 
then,  in  order  to  bring  about  a  preponderating  influence  of  the  barium 
sulphate  over  the  tendency  to  remain  in  solution  and  so  allow  it  to 
form  clumps.  If  we  heat  serum  diluted  in  salt  solution  for  one- 
quarter  hour  to  different  temperatures  (55  degrees,  70  degrees, 
85  degrees  and  100  degrees),  we  find  that  an  agglutination 
of  barium  sulphate  is  produced  by  serum  heated  to  85  or  to  100 
degrees,  but  that  dissociation  is  produced  by  serum  heated  to  55 
or  to  70  degrees.  It  is  to  be  noted  that  serum  heated  to  70  degrees 
has  not  whitened  perceptibly,  whereas  serum  heated  to  85  degrees 
has  become  milky. 

These  facts  all  go  to  show  that  in  a  mixture  of  barium  sulphate 
and  serum  either  an  agglutination  or  dissociation  is  produced,  accord- 
ing to  the  more  or  less  marked  colloidal  condition  of  the  serum 
employed.  These  two  very  different  phenomena,  however,  depend 
on  the  same  fact.  The  adhesion  of  the  colloids  to  the  suspensions 
is  just  the  same  as  the  union  between  the  suspension  and  blood 
corpuscles.  The  agglutination  of  corpuscles  by  chemical  pre- 
cipitates and  the  suspension  of  these  precipitates  in  serum  have  one 
common  explanation,  although  their  appearance  is  quite  different. 

It  would  seem  to  us  that  the  existence  of  an  adhesion  between 
serum  and  a  suspension  like  barium  sulphate  explains  the  inhibiting 
effect  that  the  serum  plays  in  the  agglutination  of  corpuscles  by 
means  of  suspensions.  This  adhesion  is  the  result,  evidently,  of  an 
affinity  between  the  suspension  and  serum,  and  we  need  only  imagine 
that  this  affinity  is  stronger  than  the  one  between  the  corpuscles 
and  the  suspension.  The  suspension,  then,  would  choose  to  unite 
with  the  serum  rather  than  with  the  corpuscles. 

Up  to  the  present  point  we  have  simply  referred  to  the  experi- 
ments with  suspensions,  corpuscles  and  serum  and  suggested  the 
conclusions  drawn  from  the  data  obtained.  We  should  like  now  to 


330  STUDIES  IN   IMMUNITY. 

glance  for  a  moment  at  the  phenomena  observed  in  the  study  of 
colloidal  substances  in  the  light  of  what  we  have  learned  from  our 
study  of  the  suspensions,  even  though  such  a  consideration  must 
be  largely  hypothetical.  Henri,  .Lalou,  Mayer,  and  Stodel  found 
that,  on  mixing  a  stable  negative  colloid,  that  is  to  say,  one  that  was 
little  affected  by  electrolytes,  with  a  colloid  of  the  same  charge 
that  is  unstable,  that  is  to  say,  very  susceptible  to  salts,  there  is  no 
precipitation  and  the  mixture  becomes  even  more  insusceptible 
to  electrolytes  than  is  the  unstable  colloid.  In  a  precipitate  caused 
by  a  flocculation  from  electrolytes  both  colloids  are  present.  Un- 
fortunately this  fact  gives  no  indication  of  the  relation  between  the 
two  coUoids  while  in  solution.  One  is  tempted  to  suppose  that  the 
stable  colloid  protects  the  unstable  colloid  from  the  electrolytes 
owing  to  an  adhesion  between  their  particles.  Such  protection 
of  an  unstable  colloid  by  a  stable  colloid  evidently  resembles  the 
protection  of  barium  sulphate  against  gravity  by  serum.  We  have 
already  shown  that  this  protection,  which  leads  to  a  suspension 
of  the  sulphate,  is  due  to  an  adhesion  between  this  substance  and 
the  colloids  of  the  serum.  It  would  therefore  seem  to  us  not 
unreasonable  to  suppose  that,  in  mixtures  with  the  same  electric 
charge,  the  protection  of  the  unstable  by  the  stable  colloid  is  due 
to  an  adhesion  between  the  particles  of  the  two  substances.  There 
would  be,  then,  an  adhesion,  as  in  mixtures  of  two  colloids  of  opposite 
charges;  but  whereas,  in  this  latter  case,  a  flocculation  occurs,  no 
such  result  happens  in  mixtures  of  colloids  with  the  same  charge. 
An  adhesion  thus  would  seem  to  take  place  between  colloids  with- 
out any  relation  to  their  electric  charge.  Flocculation  subsequently 
does  or  does  not  occur,  according  to  the  case  under  consideration. 
This  flocculation,  however,  is  not  indispensable  as  a  proof  of 
adhesion  between  the  two  colloids  and  its  absence  cannot  be  inter- 
preted as  a  certain  indication  of  an  absence  of  adhesion.  The 
precipitation  of  these  colloids  would  seem  due  to  factors  some  of 
which  are  already  known  (electrolytes  and  contrary  electric 
charges). 

The  existence  of  such  an  adhesion  would  readily  explain  a  phe- 
nomenon that  has  been  observed  by  Mangin  and  Henri  which  deals 
with  the  opposition  to  agglutination  of  corpuscles  by  means  of 
colloids,  both  negative  and  positive,  produced  by  small  amounts 


AGGLUTINATION  OF  RED  BLOOD  CELLS.  331 

of  serum.*  If  we  consider  the  serum  as  a  negative  colloid,  it  is 
easily  understandable  how  it  opposes  the  action  of  positive  col- 
loids on  corpuscles;  it  flocks  these  colloids  as  any  negative  colloid 
flocks  a  positive  one.  This  explanation,  however,  according  to 
Mangin  and  Henri,  is  not  applicable  in  the  case  of  negative  colloids, 
since  the  latter  are  not  precipitated  by  serum.  These  authors  have 
explained  the  inhibiting  effect  of  serum  on  the  agglutinating  property 
of  negative  colloids  as  due  to  the  opposition  that  the  stable  colloid 
has  on  the  flocculation  of  unstable  colloids  of  the  same  charge  by 
means  of  electrolytes,  which,  in  the  case  in  point,  are  the  intra- 
corpuscular  salts  diffused  in  the  fluid.  As  we  have  already  seen, 
this  interpretation  is  based  on  the  explanation  that  they  have 
given  of  the  agglutination  of  red  blood  cells  by  means  of  colloids, 
with  which  we  have  already  dealt. 

If  the  hypothesis  of  adhesion  between  stable  colloids  (serum) 
and  unstable  colloids  of  the  same  electric  charge  is  exact,  it  would 
suffice,  in  explaining  the  inhibition  of  agglutination  of  corpuscles 
with  negative  unstable  colloids  by  serum,  to  consider  that  the 
affinity  of  the  colloids  for  serum  agrees  with  their  affinity  for  cor- 
puscles. The  inhibition  or  agglutination  of  red  blood  cells  by 
serum,  and  the  flocculation  of  positive  colloids  by  serum,  and  the 
non-flocculation  of  negative  colloids  and  the  dissociation  of  barium 
sulphate  would  all  be  due  to  the  same  cause. 


CONCLUSIONS. 

1.  Certain  chemical  precipitates  agglutinate  and  then  hemolyze 
washed  red  blood  cells. 

2.  This  agglutination  is  due  to  the  direct  action  of  these  precip- 
itates and  corpuscles  upon  one  another. 

3.  It  is  probable  that  the  agglutinating   property  of  colloids 
for  corpuscles  is  due  fundamentally  to  a  direct  interaction  of  these 
two  substances. 

4.  Serum  even  in  small  doses  prevents  the  agglutination  and 
hemolysis  of  corpuscles  by  precipitates. 

*  This  is  evidently  analogous  with  the  fact  that  we  have  referred  to  in  our  first 
article,  namely,  that  serum  inhibits  the  agglutination  and  hemolysis  of  red  blood 
cells  by  suspensions. 


332  STUDIES  IN  IMMUNITY. 

5.  Fresh  serum  retains  in  suspension  certain  fine  precipitates, 
such  as  the  sulphate  of  barium. 

6.  This  dissociation  of  barium  by  serum  is  due  to  an  adhesion 
between  this  substance  and  the  albuminous  colloids  of  the  serum, 
and  is  similar,  therefore,  to  the  agglutination  of  barium  sulphate  by 
corpuscles,  in  that  they  are  both  due  to  the  adhesion  of  particles 
in  suspension  to  the  precipitate.    The  adhesion  of  the  albuminous 
substance  of  serum  with  suspensions  they  have  dissociated  is  easily 
demonstrable  by  the  fact  that  suspensions  dissociated  by  serum 
remain  so  when  the  excess  of  serum  is  removed  and  also  by  the 
fact  that  with  certain  precipitates  the  colloidal  substances  that 
have  been  attached  to  them  and  have  kept  them  in  suspension  may 
be  liberated  by  dissolving  the  precipitates  (tricalcium  phosphate). 

7.  The  intensity   of   the    tendency   with   which   the   particles 
attached  to  the  suspension  tend  to  remain  in  suspension  plays  a 
distinct  role  in  the  appearance  of  an  agglutination  or  of  a  dissociation 
of  the  precipitate. 

8.  It  is  possible  that,  in  a  mixture  of  two  colloids  of  the  same 
electric  sign,  one  stable  and  the  other  unstable,  the  protection  of 
the  first  for  the  second  against  the  flocculating  action  of  electro- 
lytes is  due  to  a  reciprocal  adhesion  of  the  particles  of  the  colloids. 


XVI.    OBSERVATIONS    ON    THE    SINGLE    NATURE   OF 
HEMOLYTIC  IMMUNE  BODIES,  AND  ON  THE  EXIS- 
TENCE OF  SO-CALLED  "COMPLEMENTOIDS."* 

BY  FREDERICK  P.   GAY,  M.  D. 

This  article  deals  primarily  with  two  hemolytic  phenomena,  by 
means  of  which  two  hypotheses  have  apparently  been  proved 
experimentally.  Although  I  have  dealt  in  each  instance  with  the 
classical  example  only,  my  results  are  doubtless  applicable  to 
hemolysis  in  general. 

I.  THE  NATURE  OF  A  HEMOLYTIC  IMMUNE  BODY  AS  REGARDS  ITS 
REACTIVATING  SERA. 

As  Bordetf  was  first  to  show,  hemolysis  of  rabbit  red  blood  cor- 
puscles may  be  accomplished,  in  the  presence  of  a  suitable  heated 
immune  body  (sensibilisatrice),  by  any  one  of  several  alexins,  even 
by  rabbit  alexin ;  in  this  latter  case,  however,  the  amount  of  immune 
body  necessary  to  produce  hemolysis  is  sensibly  increased  over  the 
amount  necessary  when  guinea-pig  alexin  is  used.  Ehrlich  and 
MorgenrothJ  were  able  to  corroborate  this  observation  and  also 
to  add  several  analogous  instances  which  show  clearly  that  the 
relation  of  such  immune  body  dosage  is  a  variable  one.  The 
explanation  which  they  offer,  in  harmony  with  their  "Lateral- 
chain  theory,"  is  briefly  as  follows:  In  a  given  case  the  amount 
of  heated  serum  of  a  guinea-pig  immunized  against  rabbit  corpuscles 
necessary  to  hemolyze  a  given  dose  of  rabbit  washed  corpuscles  is 
ten  times  as  great  if  rabbit  "complement"  (fresh  serum)  is  used  to 
reactivate,  as  when  guinea-pig  "complement"  is  used.  This  is  due 
to  the  fact  that  a  given  immune  serum  contains  a  series  of  "partial 
immune  bodies,"  all  active  against  the  specific  corpuscles,  but 

*  Centralbl.  f.  Bakt.,  etc.,  I.  Abt.  Originate.  Bd.  XXXIX,  1905,  p.  172. 
f  Bordet,  see  p.  134. 

J  Ehrlich  and  Morgenroth,  See  Collected  Studies  on  Immunity,  Ehrlich- 
Bolduan,  John  Wiley  &  Sons,  p.  67. 

333 


334  STUDIES  IN   IMMUNITY. 

having  each  an  affinity  for  one  certain  "complement."  By  natu- 
ral law,  only  the  smallest  number  of  such  bodies  in  serum  would 
have  "  complementophilic  "  arms  suitable  to  join  with  the  proper 
" complement"  of  the  corpuscles  to  be  destroyed.  In  the  given 
case  a  hemolytic  dose  of  the  guinea-pig  >  rabbit  immune  serum  with 
guinea-pig  " complement"  may  be  represented  by  the  formula  1  A 
+  T^  B,  "A"  being  partial  immune  bodies  joining  only  with  the 
guinea-pig  complement,  "B"  being  partial  immune  bodies  joining 
only  with  the  rabbit  complement.  In  order  to  obtain  hemolysis 
with  rabbit  complement  it  is  necessary,  logically,  to  have  ten  such 
doses  (i.e.,  10  A  +  1  B)  in  order  to  have  one  whole  dose  of  "B." 
Ehrlich  and  Morgenroth*  have  sought  further  to  establish  this 
hypothesis  of  the  multiplicity  of  immune  bodies  in  a  given  immune 
serum  by  means  of  certain  experiments  with  anti-immune  bodies 
—  a  subject  which  we  need  not  here  consider. 

After  my  first  results  on  the  subject  I  found  that  this  interesting 
question  had  already  been  considered  in  part  by  Muir  and  Brown- 
ing,! wno  nave  come  to  practically  the  same  conclusions  as  myself, 
although  they  were  unable  wholly  to  refute  the  hypothesis  of  the 
multiplicity  of  immune  bodies  on  account  of  certain  technical  diffi- 
culties; these  difficulties  I  have  been  able  to  obviate  by  a  specially 
devised  method. 

Although  nowhere  expressly  stated  by  Bordet  or  by  Ehrlich  and 
Morgenroth,  I  have  found,  in  common  with  Muir  and  Browning, 
that,  when  we  reactivate  sensitized  rabbit  corpuscles  (heated  guinea- 
pig  >  rabbit  immune  serum)  with  rabbit  alexin,  not  only  is  the 
requisite  dose  of  immune  body  large,  but  also  the  dose  of  rabbit 
alexin  relatively  high.  The  relative  amounts  of  the  immune  serum 
necessary  in  the  presence  of  sufficient  alexin  of  either  animal,  and 
the  relative  amounts  of  each  alexin  when  sufficient  immune  body 
is  present  are  so  well  shown  in  the  tables  of  Muir  and  Browning 
that  I  omit  similar  examples.  As  regards  the  relatively  high  dose 
of  rabbit  alexin  against  its  own  corpuscles  the  following  (Table  I) 
shows  that  it  takes  nearly  three  times  as  much  alexin  to  destroy 
numerically  a  smaller  number  of  its  own  sensitized  corpuscles  than 
of  sensitized  ox  corpuscles. 

*  Ehrlich  and  Morgenroth,  See  Collected  Studies  on  Immunity,  Ehrlich- 
Bolduan,  John  Wiley  &  Sons,  p.  101. 

t  Muir  and  Browning,  Proceed.  Royal.   Soc.,  Vol.  LXXIV,  1904,  p.  298. 


SINGLE  NATURE  OF  HEMOLYTIC  IMMUNE  BODIES.         335 


TABLE  I. 

Red  blood  corpuscles  of  ox  per  c.mm. ,  5,880,000.     Red  blood  corpuscles  of  rabbit  per  c.mm., 

4,240,000. 


No. 

Washed  rabbit  cor- 
puscles. 

S.  guinea-pig  >  rabbit, 
55°. 

Rabbit  alexin. 

Hemolysis. 

1 
2 
3 
4 
5 

0.025  c.c. 
0.025  c.c. 
0.025  c.c. 
0.025  c.c. 
0.025  c.c. 

0.25  c.c 
0.25  c.c. 
0.25  c.c. 
0.25  c.c 
0.25  c.c. 

0.2       c.c. 
0.15     c.c. 
0.1       c.c. 
0.075  c.c. 
0.05    c.c. 

0.075  c.c. 
0.05    c.c 

complete 
partial 
slight 
slight 
slight 

complete 
slight 

Washed  ox  corpuscles. 

S.'  rabbit  >  ox,  55°. 

0.025  c.c. 
0.025  c.c. 

0.15  cc 
.  0.15  c.c. 

As  regards  the  technic  of  hemolytic  experiments,  it  may  be  well 
to  note  the  role  played  by  an  excessive  amount  of  isotonic  salt 
solution  in  the  suspensions  of  test  corpuscles  employed  by  many 
observers.  It  would  seem,  a  priori,  desirable  to  deal  with  cor- 
puscles as  nearly  as  possible  in  their  normal  condition  of  suspension 
in  the  blood,  but  the  advantages  of  a  5  per  cent  suspension  are 
manifest,  since  smaller  amounts  can  be  accurately  measured  and  a 
great  waste  of  often  precious  materials  obviated.  But  I  have  noted 
repeatedly  that  the  dilution  of  a  small  amount  of  alexic  serum  by 
so  great  an  excess  of  isotonic  solution  unquestionably  affects  its 
activity  and  often  completely  inhibits  it.  This  is  well  shown  in  the 
case  of  sensitized  rabbit  corpuscles  when  acted  on  by  rabbit  alexin. 

TABLE  II. 


Washed  rabbit  corpuscles. 

S.  guinea-pig  > 
rabbit,  55°. 

Rabbit  alexin. 

Hemolysis. 

5%  suspension  in 
NaCl  (0.85%). 

Centrifugalized 
deposit  of  5%  sus- 
pension. 

2  C.C. 
2  C.C. 

=  15  C-C. 

=  T<5  c.c. 

0.4  c.c. 
0.4  c-c. 
0.4  c.c. 
0.4  c.c. 

0.4  c.c. 
0.4  c.c. 
1.2  c.c. 
1.2  c.c. 

none 
partial 
slight 
complete 

336  STUDIES  IN  IMMUNITY. 

A  similar  and  yet  more  striking  example  of  the  effect  of  a  great 
excess  of  isotonic  solution  will  be  given  later.  To  retain  the  use- 
fulness of  the  5  per  cent  suspension  and  to  obviate  its  errors,  I  have 
employed  this  method  of  centrifugalizing  the  suspension  in  the 
actual  experimental  tube,  removing  the  supernatant  fluid,  and 
subsequently  adding  the  small  amount  of  physiological  solution 
necessary  to  bring  the  blood  suspension  to  its  primitive  volume. 

Another  source  of  possible  error  in  experiments  of  this  nature 
is  the  presence  of  anti-alexic  action  in  the  excess  of  heated  immune 
serum,  which,  in  rare  instances,  inhibits  the  activity  of  the  proper 
alexin  of  the  corpuscles.  The  occurrence  of  such  an  action  is  not 
surprising  when  we  consider  that  the  usual  procedure  for  the  pro- 
duction of  an  immune  serum  is  to  immunize  animals  with  the  whole 
defibrinated  blood,  that  is,  with  corpuscles  plus  serum.  The  remedy, 
of  course,  consists  in  removing  the  excess  of  immune  serum  after 
it  has  been  allowed  to  come  in  contact  with  the  corpuscles. 

Although  the  amount  of  rabbit  alexin  necessary  to  hemolyze 
sensitized  rabbit  corpuscles  is  relatively  large,  it  is  easy  to  show 
that  smaller  doses  combine  perfectly  with  the  corpuscle-immune 
body  complex.  As  Muir  expresses  it,  the  combining  power  is  per- 
fect although  the  toxic  power  is  slight.  Such  variations  in  toxicity 
were  previously  noted  by  Bordet  *  in  consonance  with  his  quanti- 
tative idea  of  the  difference  in  alexins. 

If  we  take  Table  I,  for  example,  and  add  to  each  of  the  super- 
natant fluids  of  tubes  1  to  5,  in  which  more  or  less  hemolysis  has 
occurred,  sensitized  ox  corpuscles  (ox  corpuscles,  0.025  of  a  cubic 
centimeter  +  S.  rabbit  >  ox,  55  degrees,  0.05  of  a  cubic  centimeter), 
we  obtain  no  trace  of  hemolysis  in  any  tube,  which  shows  that  the 
rabbit  alexin  has  already  combined  perfectly  with  the  sensitized 
rabbit  corpuscles. 

Before  proceeding  to  the  proof  of  the  simple  nature  of  the  guinea- 
pig-rabbit  immune  body,  it  will  be  necessary  to  describe  an  experi- 
ment which  offers  a  valuable  technical  method  for  the  solution  of 
this  problem,  and  which  may  also  prove  of  service  in  other  hemolytic 
experiments.  It  is  already  known  that  when  corpuscles  have  been 
hemolyzed  by  means  of  a  heated  specific  serum,  and  a  dose  of 
alexin,  which  is  not  in  excess,  added,  the  resulting  stromata  have 

*  Bordet,  see  p.  234. 


SINGLE  NATURE  OF  HEMOLYTIC  IMMUNE  BODIES.         337 

still  the  ability  to  absorb  additional  portions  of  alexin.  I  have 
found  also  that  stromata  produced  by  heating  corpuscles  to  55 
degrees  for  half  an  hour  retain  unimpaired  their  power  of  absorbing 
immune  body,  or,  if  previously  sensitized,  of  absorbing  alexin. 
Rabbit  corpuscles  are  completely  hemolyzed  by  this  temperature, 
but  ox  corpuscles,  although  showing  a  reduction  of  hemoglobin, 
remain,  for  the  most  part,  microscopically  intact.  The  affinities 
of  ox  corpuscles  after  heating  to  55  degrees  are  shown  by  the  follow- 
ing experiment: 

THREE  TUBES  ARE  PREPARED  AS  FOLLOWS: 

Tube  A.     Ox  corpuscles  previously  heated  to  55  degrees  (not  hemo- 
lyzed) 0.2  c.c. 
Serum  rabbit  >  ox,  55  degrees  0.8  c.c. 
S.  guinea-pig  (alexin)  0.5  c.c. 
Tube  B.     Ox  corpuscles,  55  degrees  0.2  c.c. 
S.  normal  rabbit,  55  degrees  0.8  c.c. 
S.  guinea-pig  0.5  c.c. 
Tube  C.     Ox    corpuscles,    0.2  +  Serum    rabbit  >  ox,   0.8,   heated 

to  55  degrees 

S.  guinea-pig  0.5  c.c. 

In  tubes  A  and  C  hemolysis  is  complete;  in  B  nul. 

To  the  centrifugalized  supernatant  fluids  of  A,  B  and  C  is  added 
subsequently 

Ox  corpuscles  0.1  c.c. 

S.  rabbit  >  ox,  55  degrees  0.4  c.c. 

Tube  D  (control).     Ox  corpuscles  0.1  c.c. 

S.  rabbit  >  ox,  55  degrees  0.4  c.c. 

S.  guinea-pig  0.2  c.c. 
In  tubes  B  and  D  hemolysis  is  complete. 
In  tubes  A  and  C  hemolysis  nul. 

Similar  results  may  be  obtained  with  rabbit  corpuscles  with  the 
exception  that  they  are  hemolyzed  by  heating  to  55  degrees. 

In  considering  the  nature  of  the  guinea-pig  >  rabbit  immune  body 
I  shall  give  an  example  in  which  the  relative  doses  of  the  immune 
body  and  the  alexin  of  rabbit  or  of  guinea-pig  chanced  to  be  the 
same  as  in  the  classical  example  of  Ehrlich.  Of  course  it  is  under- 
stood that  this  relation  is  not  a  fixed  one,  as  Ehrlich  has  shown.  In 
this  case,  in  order  to  produce  hemolysis  of  rabbit  corpuscles  in  the 
presence  of  sufficient  rabbit  alexin,  it  took  ten  times  the  amount  of 
heated  immune  serum  as  when  using  guinea-pig  alexin.  In  the 
language  of  the  Ehrlich  hypothesis,  the  necessary  dose  of  immune 
serum  with  rabbit  alexin  may  be  represented  by  10  "A"  +  1  "B," 
"A"  being  the  partial  immune  body  suitable  for  guinea-pig  alexin 


338  STUDIES  IN   IMMUNITY. 

and  "  B  "  the  partial  immune  body  for  rabbit  alexin.  Rabbit  alexin 
cannot  join  "A"  or  hemolysis  would  be  produced  with  the  same 
dose  of  immune  body  as  when  employing  guinea-pig  alexin.  Now, 
if  we  produce  hemolysis  with  rabbit  alexin  in  the  presence  of  the 
necessary  amount  of  immune  serum,  the  1  "B"  will  be  satisfied, 
that  is,  one-eleventh  only  of  the  amboceptors  will  have  their  "com- 
plementophilic  "  arms  plugged,  while  10  "A,"  or  the  remaining  ten- 
elevenths,  will  be  attached  to  the  stromata  and  be  still  ready  to  fix 
many  hemolytic  doses  of  guinea-pig  alexin.  This  is  the  logic  of  the 
Ehrlich  hypothesis.  Experimentally  we  obtain  the  following  results : 

Rabbit  corpuscles  are  hemolyzed  with  rabbit  alexin  and  guinea- 
pig  >  rabbit  heated  immune  serum;  the  stromata  are  then  heated 
to  55  degrees,  which  will  destroy  any  rabbit  alexin  that  may  be  free, 
or  prevent  any  further  toxic  action  from  it,  but  will  leave  the  cor- 
puscle-immune-body complex  still  unaffected  as  regards  its  further 
absorption  of  the  guinea-pig  alexin  by  its  supposedly  unsatisfied 
10  "  A"  amboceptors.  In  fact,  we  find  that  the  alexin  of  the  gui- 
nea-pig subsequently  added  remains  entirely  free;  not  one  slight 
portion  of  it  has  been  attached,  as  is  shown  by  suitable  controls. 
In  other  words,  the  fixation  of  rabbit  alexin  will  completely  pre- 
vent the  subsequent  fixation  of  guinea-pig  alexin.  The  details  of 
the  experiment  follow. 

Minimal  hemolytic  dose  of  guinea-pig  >  rabbit  immune  serum,  55 
degrees,  for  0.025  of  a  cubic  centimeter  of  washed  rabbit  corpuscles. 

with  rabbit  alexin 0.25  c.c. 

with  guinea-pig  alexin 0.025  c.c. 

Relation  10  :  1. 

THREE  LARGE  TUBES  ARE  PREPARED: 

Tube  A.     Washed  rabbit  corpuscles  0.125  c.c. 

S.  guinea-pig  >  rabbit,  55  degrees  1.25  c.c. 

S.  rabbit  (alexin)  1.25  c.c. 

Tube  B.     Washed  rabbit  corpuscles  0.125  c.c. 

S.  guinea-pig  >  rabbit,  55  degrees  1.25  c.c. 

S.  rabbit,  55  degrees  1.25  c.c. 

Tube  C.     Washed  rabbit  corpuscles  0.125  c.c. 

S.  normal  guinea-pig,  55  degrees  1.25  c.c. 

S.  rabbit,  55  degrees  1.25  c.c. 

Contact  at  37°  C  for  1  hour. 
Tube  A  is  perfectly  hemolyzed. 
Tube  B,  agglutination;  no  hemolysis. 
Tube  C,  no  change. 


SINGLE  NATURE  OF   HEMOLYTIC  IMMUNE  BODIES.         339 

The  contents  of  the  three  tubes  are  then  heated  to  55  degrees  for 
one-half  an  hour.  Hemolysis  complete  in  all  tubes.  From  each  of 
the  tubes  A,  B  and  C  are  prepared  three  smaller  tubes,  each  con- 
taining 0.5  of  a  cubic  centimeter  of  the  stroma  mixture,  that  is,  the 
stromata  of  0.025  of  a  cubic  centimeter  of  rabbit  corpuscles.  To  the 
corresponding  tubes  in  each  series  is  added  the  same  amount  of 
fresh  guinea-pig  alexin,  as  follows : 

"A"  series.  Tube  1.  "A"  mixture  0.5  c.c. 

S.  guinea-pig  0.025  c.c. 

Tube  2.  "A"  mixture  0.5  c.c. 

S.  guinea-pig  0.05  c.c. 

Tube  3.  "A"  mixture  0.5  c.c. 

S.  guinea-pig  0.1  c.c. 

"B"  series.  Tube  4.  "B"  mixture  0.5  c.c. 

S.  guinea-pig  0.025  c.c. 

Tube  5.  "  B "  mixture  0.5  c.c. 

S.  guinea-pig  0.05  c.c. 

Tube  6.  "B"  mixture  0.5  c.c. 

S.  guinea-pig  0.1  c.c. 

"C"  series.  Tube  7.  "C"  mixture  0.5  c.c. 

S.  guinea-pig  0.025  c.c. 

TubeS.  "C"  mixture  0.5  c.c. 

S.  guinea-pig  0.05  c.c. 

Tube  9.  "C"  mixture  0.5  c.c. 

S.  guinea-pig  0.1  c.c. 

Contact  at  37°  C  for  1  hour.  Tubes  centrifugalized.  The  supernatant  fluid 
of  each  tube  is  placed  in  a  new  tube  containing: 

Washed  rabbit  corpuscles  0.025  c.c. 

S.  guinea-pig  >  rabbit,  55  degrees  0.05    c.c. 

Resultant  hemolysis  is  as  follows: 

"A"  series  "B"  series  "C"  series 

1.  Marked  4.   None  7.   Marked 

2.  Complete  5.   None  8.   Complete 

3.  Complete  6.   None  9.   Complete 

In  series  "A"  the  guinea-pig  alexin  is  left  entirely  free  because 
the  avidity  of  the  corpuscle-immune-body  complex  for  alexin  has 
been  entirely  satisfied  by  the  rabbit  alexin.  In  series  "B"  the 
sensitized  corpuscles,  subjected  to  the  same  conditions  of  heat  and 
serum  dilution  as  series  "A,"  absorb  perfectly  the  guinea-pig  alexin. 
In  series  "C"  we  have  a  control,  with  no  immune  serum,  which 
leaves  just  as  much  but  no  more  guinea-pig  alexin  free  than  does 
series  "A. "  The  certain  conclusion  from  this  experiment  is  that 
the  immune  body  against  rabbit  corpuscles  is  of  a  simple  nature  as 
regards  combination  with  one  or  another  alexin. 


340  STUDIES  IN  IMMUNITY. 

II.   THE  NATURE  OF  A  SO-CALLED  "COMPLEMENTOID." 

In  his  analogy  between  antitoxic  and  hemolytic  sera  Ehrlich 
presupposed  the  existence  of  substances  called  "  complementoids " 
—derived  from  " complements/'  as  "toxoids"  are  supposed  to  be 
derived  from  toxins.  These  "complementoids"  are  depicted  as 
possessing  a  "haptophore  group"  but  no  "zy  mo  toxic"  group,  in 
distinction  from  complements,  which  have  both.  That  is,  they 
have  none  of  the  toxic  or  hemolytic  activity  of  complements,  but 
may  give  rise,  when  injected,  to  "  anticomplements,"  and  may,  under 
certain  conditions,  combine  with  amboceptors.  After  the  hypothe- 
sis followed  apparent  experimental  proof  of  the  existence  of  such 
bodies.  Ehrlich  and  Sachs*  described  a  phenomenon  which  occurs 
in  dog  serum  which  they  call  "Verstopfung  der  Ambozeptoren 
durch  Komplementoide, "  -the  plugging  of  amboceptors  by 
means  of  complementoids.  The  details  are  briefly  as  follows:  Fresh 
dog  serum  hemolyzes  guinea-pig  corpuscles.  Dog  serum  heated 
to  51°  C  for  one-half  hour  loses  its  power  to  produce  this  hemolysis 
owing  to  the  destruction  at  this  temperature  of  the  very  labile 
complement.  This  heated  serum  (Ambozeptor)  may  be  reactivated 
by  guinea-pig  ''complement."  If,  however,  the  corpuscles  are  left 
in  contact  with  the  heated  dog  serum  at  37°  C  for  2  hours  the 
subsequent  addition  of  fresh  guinea-pig  serum  produces  no  hemoly- 
sis. f  This  fact  is  ascribed  to  a  plugging  of  the  "  complementophilic 
arm"  of  the  dog  amboceptor  by  means  of  the  " complementoids " 
present  in  the  serum  heated  to  51  degrees.  The  amboceptor  is  said 
to  be  destroyed  by  a  temperature  of  60  degrees;  the  somewhat 
unusual  nature  of  this  amboceptor  is  fully  discussed  in  another 
place  by  Sachs,:):  but  need  not  concern  us  here. 

*  Ehrlich  and  Sachs,  See  Collected  Studies  on  Immunity,  Ehrlich-Bolduan, 
John  Wiley  &  Sons,  p.  209. 

f  Although  experimental  proof  that  the  subsequently  added  alexin  has  not 
been  combined  is  not  offered  by  the  authors,  such  a  matter  is  easy  to  determine. 
If  we  centrif  ugalize  such  a  tube  and  to  the  supernatant  fluid  add  heated  dog  serum, 
and  then  corpuscles;  hemolysis  is  produced,  showing  that  the  alexin  has  really 
been  kept  out  of  combination  by  the  heated  dog  serum  united  to  the  corpuscles. 
The  control  is  a  tube  in  which  hemolysis  has  occurred  by  the  addition  of  dog  serum 
and  alexin  at  the  same  time,  in  which  case  no  subsequent  alexic  activity  is 
detectable. 

t  Sachs,  H.,  See  Collected  Studies  on  Immunity,  Ehrlich-Bolduan,  John 
Wiley  &  Sons,  idem,  p.  186. 


SINGLE  NATURE  OF  HEMOLYTIC  IMMUNE  BODIES.        341 

In  dealing  with  this  phenomenon,  I  have  found  the  results  as 
given  do  not  express  the  entire  truth  as  I  have  found  it.  The 
doses  given  in  the  "plugging"  experiment  described,  are  as  follows: 

Guinea-pig  washed  corpuscles,  5  per  cent  suspension  1  c.c. 

Dog  serum,  50  to  51  degrees  0.5  c  c. 

Guinea-pig  serum  0.5  c.c. 

which  doses  I  shall  use  in  the  experiments  to  be  described.  I  have 
found,  as  a  matter  of  fact,  that  dog  serum  heated  to  the  degree 
mentioned  does  always  give  a  slight  hemolysis  of  guinea-pig  cor- 
puscles, a  fact  which  is  masked  by  the  excess  of  salt  solution  used 
in  the  5  per  cent  suspension.  If  we  deal  with  the  centrifugalized  de- 
posit of  1  c.c.  of  a  5  per  cent  suspension,  or  simply  with  0.05  of  a  cubic 
centimeter  of  washed  corpuscles,  this  hemolysis  is  clearly  shown. 
That  dog  serum  heated  for  one-half  hour  to  50  degrees,  51  degrees 
or  52  degrees  does  possess  distinct  hemolytic  power  for  guinea-pig 
corpuscles  may  be  shown  more  distinctly  in  the  following  way: 
If  we  prepare  a  series  of  tubes  each  containing  1  c.c.  of  suspensions 
of  corpuscles  of  5  per  cent,  3  per  cent,  1  per  cent  and  one-half  per 
cent  respectively,  and  to  each  tube  add  the  given  dose  of  heated  dog 
serum  (0.5  of  a  cubic  centimeter,  50  to  51  degrees),  we  find  after  2 
hours  at  37°  C  that  in  the  tubes  at  1  per  cent  and  one-half  per  cent 
hemolysis  is  complete;  or,  better  still,  if  we  use  the  centrifugalized 
deposits  of  such  a  series  of  suspensions  without  the  excess  of  salt 
solution,  we  find  that  hemolysis  is  complete  in  the  tubes  at  3  per 
cent,  1  per  cent  and  one-half  per  cent  and  marked  in  the  5  per  cent 
tube.  It  is  evident  from  such  an  experiment  that  the  so-called 
"  complementoid  "  is  nothing  more  than  a  "  complement"  the  hemo- 
lytic activity  of  which  has  been  impaired,  but  not  destroyed,  by  the 
heating.  That  the  subsequent  addition  of  guinea-pig  serum  does 
not  sensibly  increase  the  hemolysis  in  such  a  series  of  tubes  is  per- 
fectly true,  which  means  that  there  is  a  "plugging"  (Verstopfung) 
by  means  of  the  partially  destroyed  "complement. "  The  combining 
power  of  the  "complement"  has  been  no  more  destroyed  than  the 
toxic  power.  If,  instead  of  dog  serum  heated  to  51  degrees,  we  use 
dog  serum  heated  to  56  degrees,  we  find  another  more  striking  exam- 
ple of  this  modified  "complement. "  To  a  similar  series  of  tubes  at 
5  per  cent,  3  per  cent,  1  per  cent  and  one-half  per  cent  respectively 
add  0.5  of  a  cubic  centimeter  of  dog  serum  heated  to  56  degrees  for 


342 


STUDIES   IN   IMMUNITY. 


one-half  hour.  After  3  hours  at  37°C  slight  hemolysis  is  present  only 
in  the  tube  at  one-half  per  cent  (complete  hemolysis  if  the  deposit  of 
such  a  suspension  is  used),  which  shows  that  not  even  at  this  tem- 
perature is  the  toxic  action  of  the  "complement"  entirely  destroyed. 
If  we  add  0.5  of  a  cubic  centimeter  of  fresh  guinea-pig  serum  to  an 
analogous  series  of  tubes,  it  is  found  that  hemolysis  is  all  but  com- 
plete throughout  the  series,  that  is,  only  the  slightest  " plugging" 
has  occurred.  The  combining  ability  of  the  dog  complement  is 
impaired  equally  with  and  proportionately  to  the  toxic  ability. 
The  tables  follow: 

TABLE   I. 


Washed 

guinea-pig 
corpuscles  in 

Dog  serum, 
50-51°  C. 

Contact,  37°. 

NaCl  sol. 

Contact,    37°. 

Hemolysis. 

suspension  of 

5%  1  c-c. 

0.5  C  c. 

2  hours 

0.5  c.c. 

1  hour 

trace 

3%  1  c  c. 

0.5  c  c. 

2  hours 

0.5  c.c. 

1  hour 

marked 

1%  1  c  c. 

0.5  c  c. 

2  hours 

0.5  c.c. 

1  hour 

complete 

0.5%  Ic.c. 

05  c-c- 

2  hours 

0.5  c-c 

1  hour 

complete 

TABLE   II. 


Deposit  of  1  c.c. 
G.  p.  corpuscles; 

Dog  serum, 
50   51°  C 

Contact,  37°. 

NaCl  sol. 

Contact,  37°. 

Hemolysis. 

suspension  of 

5%  (.05  c.c.*) 

0.5  c.c. 

2  hours 

0.5  c.c. 

1  hour 

nearly  com- 

plete 

3%  (.05  c.c.) 

0.5  c.c. 

2  hours 

0.5  c.c. 

1  hour 

complete 

1%  (.05  c.c.) 

0.5  c.c. 

2  hours 

0.5  c.c. 

1  hour 

complete 

0.5%  (.05  cc.) 

0.5  c.c. 

2  hours 

0.5  c.c. 

1  hour 

complete 

Represents  the  actual  volume  present. 
TABLE    III. 


Washed 

guinea-pig 
corpuscles  in 
suspension  of 

Dog  serum, 
50-51°  C. 

Contact,  37°. 

A  lex  in  guinea- 
Pig. 

Contact,  37°. 

Hemolysis. 

5%  1  c  c 
3%  Ice 
1%  1  cc 
0.5%  Ice. 

0.5  C.C 
0.5  c.c 
0.5  c  c 
0.5  cc 

2  hours 
2  hours 
2  hours 
2  hours 

0.5  cc 
0.5  c  c 
0.5  c  c 
0.5  c  c 

1  hour 
1  hour 
1  hour 
1  hour 

trace 
marked 
complete 
complete 

SINGLE  NATURE  OF  HEMOLYTIC  IMMUNE  BODIES. 


343 


TABLE   IV. 


Deposit  of  1 

c.c.  G.  p. 

corpuscles; 

Dog  serum, 
50-51°  C. 

Contact,  37°. 

Alexinguinea- 
pig- 

Contact,  37°. 

Hemolysis. 

suspension  of 

5%  (.05  c.c.) 

0.5  c.c 

2  hours 

0.5  cc 

1  hour 

nearly  com- 

plete 

3%  (.05  c.c.) 

0.5  c  c. 

2  hours 

0.5  c  c. 

1  hour 

complete 

1%  (.05  c.c  ) 

0.5  c  c 

2  hours 

0.5  c  c 

1  hour 

complete 

0.5%  (.05  c.c.) 

0.5c  c 

2  hours 

0.5  c  c. 

1  hour 

complete 

TABLE   V 


Washed 

guinea-pig 
corpuscles  in 
suspension  of 

Dog  serum, 
56°  C. 

Contact,  37°. 

NaCl  sol. 

Contact,  37°. 

Hemolysis. 

5%  1  c.c. 
3%  1  c.c. 
1%  1  c.c. 
0.5%  1  c.c. 

0.5  c  c 
0.5  c.c 
0.5  c  c 
0.5  c  c. 

2  hours 
2  hours 
2  hours 
2  hours 

0.5  c.c. 
0.5  P.C. 
0.5  c.c. 
0.5  c.c. 

1  hour 
1  hour 
1  hour 
1  hour 

none 
none 
none 
marked 

TABLE   VI. 


Deposit  of  1 

c.c.  G.  p.  cor- 
puscles ;     sus- 

Dog serum, 
56°  C. 

Contact,  37°. 

NaCI  sol. 

Contact,  37°. 

Hemolysis. 

pension  of 

5%  (.05  c.c.) 

0.5  c  c 

2  hours 

0.5  c.c. 

1  hour 

none 

3%  (.05  c.c.) 

0.5  c.c. 

2  hours 

0.5  c.c. 

1  hour 

none 

1%  (.05  c.c.) 

0.5  c.c. 

2  hours 

0.5  c.c 

1  hour 

slight 

0.5%  (.05  c.c.) 

0.5  c.c. 

2  hours 

0.5  c.c. 

1  hour 

complete 

TABLE   VII 


Washed 

guinea-pig 
corpuscles  in 

Dog  serum, 
56°  C. 

Contact,  37°. 

A  lex  in  guinea- 

Contact,  37°. 

Hemolysis. 

suspension  of 

pig. 

5%  1  c.c. 

0.5  c  c 

2  hours 

0  5cc 

1  hour 

nearly  com- 

plete 

3%  Ic.c 

0.5  c.c 

2  hours 

0.5  c  c 

1  hour 

complete 

1%  1  c.c 

0.5  c  c 

2  hours 

05  c  c. 

1  hour 

complete 

0.5%  1  c.c. 

0.5  c  c. 

2  hours 

05  c.c 

1  hour 

complete 

344 


STUDIES  IN   IMMUNITY 


TABLE  VIII 


Deposit  of  1 

c.c.  corpus- 
cles; suspen- 

Dog serum, 
56°  C. 

Contact,  37°. 

Alexin  guinea- 
pig- 

Contact,  37°. 

Hemolysis. 

sion  of 

5%  (  05  c  c  ) 

0.5  C  C 

2  hcurs 

05  c  c. 

1  hour 

nearly  com- 

plete 

3%  (  05  c  c  ) 

0.5  c  c 

2  hours 

0.5  c  c 

1  hour 

complete 

1%  (05  c.c  ) 

0.5  c  c 

2  hours 

0.5  c  c 

1  hour 

complete 

0.5%  (  05  c  c  ) 

0.5  c.c 

2  hours 

0.5  c  c. 

1  hour 

complete 

Controls: 
G.  p.  corpus- 
cles, 5%  sus- 
pension  in 

0.85%  NaCl.  Hemolysis. 

1  c  c        S.  dog  50-51°  .0.5  c.c.   Alex,  guinea-pig  0.5  c.c.  complete  10  mins. 
1  c.c.      S  dog         56°. 0.5  c.c.   Alex,  guinea-pig  0.5  c.c.  complete  10  mins. 

In  these  experiments  we  again  see  the  disadvantage  of  employing 
a  5  per  cent  suspension  owing  to  the  inhibiting  action  of  the  great 
excess  of  salt  solution  on  a  weak  alexin  (series  I  and  II,  III  and 
IV,  etc.).  Heating  dog  serum  to  51  degrees  merely  weakens  the 
toxic  and  combining  activity  of  the  dog  alexin;  56°  C  does  not 
wholly  destroy  either  the  toxic  or  the  combining  activities  of  the 
alexin,  but  destroys  one  as  much  as  the  other.  The  normal  immune 
body  apparently  suffers  no  impairment  by  heating  to  56  degrees 
(Controls). 

CONCLUSIONS. 

1.  Suspensions  of  blood  corpuscles  for  hemolytic  experiments 
of  5  per  cent  are  disadvantageous  unless  the  excess  of  normal  saline 
solution  be  removed,  as  alexic  power  is  often  entirely  inhibited  by 
the  great  excess  of  fluid. 

2.  Heating  to  55  degrees  for  one-half  hour  does  not  affect  the 
power  of  ox  or  rabbit  corpuscles,  even  when  hemolysis  is  produced, 
to  absorb  suitable  immune  bodies  or  alexins. 

3.  A  given  immune   serum,  active   against  rabbit   corpuscles, 
contains  a  simple  immune  body  as  regards  affinity  for  various 
alexins. 

4.  So-called  "  complementoids  "  (to  judge  from  the  test  example) 
are  simply  "  complements "  (alexins)  in  which  both  the  combining 
power  and  the  hemolytic  power  are  weakened. 


SINGLE  NATURE  OF  HEMOLYTIC  IMMUNE  BODIES.        345 

(Sachs  (Central,  fur  Bakt.,  etc.,  Abt.  I,  Orig.  Bd.  XXXIX,  1905) 
has  questioned  the  experimental  accuracy  of  the  part  of  this  article 
bearing  on  complementoids.  In  a  reply  Gay  (Central,  fur  Bakt., 
XL,  1906,  695)  has  repeated  the  experiments  and  proved  that  dog 
serum  heated  to  51°  C  is  not  only  slightly  hemolytic  for  guinea-pig 
corpuscles,  but  will  hemolyze  more  markedly  either  rabbit  or  guinea- 
pig  corpuscles  that  have  been  treated  with  a  heated  immune  serum. 
The  conclusion,  then,  is  that  in  classical  examples  of  "complement- 
oids" we  have  to  deal  with  a  " complement"  the  toxicity  of  which 
has  simply  been  attenuated,  and  that  no  satisfactory  experimental 
proof  of  the  existence  of  "complementoids"  exists.  F.  P.  G.) 


XVII.      THE     FIXATION     OF    ALEXINS    BY    SPECIFIC 
SERUM  PRECIPITATES.* 

BY  FREDERICK  P.   GAY,  M.D. 

We  possess  already  a  wealth  of  experimental  detail  relative  to 
the  specific  immune  bodies  formed  in  the  sera  of  animals  injected 
either  with  simple  cells  or  with  such  complex  fluids  as  defibrinated 
blood  or  blood  serum.  Among  the  best  known  of  these  immune 
bodies  are  the  specific  hemolysins  and  bacteriolysins,  the  activities 
of  which  have  been  most  fruitfully  studied.  We  know,  for  example, 
that  a  given  hemolytic  immune  body  (substance  sensibilisatrice, 
amboceptor)  formed  after  the  injection  of  foreign  red  blood  cells 
has  two  important  properties,  namely,  the  property  of  sensitizing 
the  causative  cell  in  such  a  manner  as  to  allow  it  to  be  destroyed  by 
the  alexin  (complement)  of  various  fresh  normal  sera;  and  secondly, 
the  power,  when  it  has  formed  a  complex  with  the  causative  cell, 
of  fixing  an  alexin.  This  second  property  is  perhaps  the  more 
important,  since  the  alexin  can  often  be  shown  to  have  been  absorbed 
by  the  cell-immune-body  complex  even  when  the  cell  itself  is  not 
destroyed.  Indeed,  this  absorption  of  alexin  has  given  the  means 
of  determining  the  existence  of  sensitizing  substances  where  they 
might  not  otherwise  have  been  shown  to  exist.  Bordet  and 
Gengouj  have  shown  that  the  majority  of  antimicrobial  sera  con- 
tain "substances  sensibilisatrices "  that  absorb  alexin  in  instances 
where  destruction  of  the  specific  bacteria  is  not  produced,  ami 
GengouJ  has  further  shown  that  the  injection  of  certain  albuminoids 
may  likewise  give  rise  to  specific  sensitizing  sera  which  may  form 
with  the  causative  substances  mixtures  that  absorb  alexin. 

*  Centralblatt  fur  Bakt.,  I  Abt.,  Orig.  XXIX,  1905,  603. 
t  Bordet  and  Gengou,  see  p.  217. 
J  Gengou,  see  p.  241. 


346 


THE  FIXATION  OF  ALEXINS.  .347 

I. 

I  was  led  recently  to  consider  the  effect  that  hemolytic  immune 
serum  heated  to  55°  C,  and  then  left  in  contact  for  a  time  with  the 
specific  red  blood  corpuscles,  might  have  on  the  activity  of  an 
alexin.  It  surprised  me  to  find  that  such  a  treated  serum,  cen- 
trifugalized  long  enough  to  free  it  of  the  corpuscles,  had  the  power 
of  neutralizing  the  hemolytic  activity  of  an  added  alexin,  as  could 
be  shown  by  subsequently  introducing  sensitized  corpuscles.  In 
controlling  more  carefully  such  an  experiment  I  found  that  the 
treated  immune  serum,  although  freed  of  all  corpuscles  by  the  first 
short  centrifugalization,  would,  when  centrifugalized  a  second  time, 
show  a  slight  cloudiness  at  the  bottom  of  the  tube,  which  micro- 
scopically exhibited  the  amorphous  character  of  specific  precipi- 
tates. If  this  precipitate  was  removed  from  the  treated  serum,  no 
fixation  of  the  alexin  took  place.  It  seems,  then,  quite  evident  that 
a  specific  serum  precipitate  has  the  power  to  fix  alexin,  and  the 
causative  factors  of  such  a  precipitate  must  now  concern  us. 

Of  the  precipitate-forming  factors  in  the  experiments  to  which 
I  have  referred  the  precipitin  was  of  course  furnished  by  the  immune 
serum,  but  the  source  of  the  precipitinogen  is  not  so  evident,  since 
washed  corpuscles  and  not  native  blood  were  used.  Further  obser- 
vations showed  that  although  the  corpuscles  for  these  experiments 
had  been  washed  once  or  twice  with  a  relatively  large  amount  of 
physiological  solution,  such  washing  was  not  sufficient  to  remove 
all  the  serum  which  bathed  the  corpuscles,  and  which,  although 
markedly  diluted,  contained  the  very  small  amount  of  precipitinogen 
necessary  to  form  a  precipitate  with  the  large  amount  of  immune 
serum  present.  As  a  matter  of  fact  it  is  extremely  difficult  to  free 
blood  corpuscles  of  all  traces  of  serum,  as  is  clearly  shown  by  some 
recent  experiments  of  Gengou,  to  whom  I  am  indebted  for  the  follow- 
ing unpublished  observations :  A  four  per  cent  alcoholic  solution  of 
mastic,  forms,  on  the  addition  of  distilled  water  in  the  proportions 
of  nine  parts  of  water  to  one  of  mastic,  an  emulsion,  which  affords 
a  delicate  reagent  for  the  albuminoids  of  blood  serum.  One  drop 
of  normal  salt  solution  containing  a  trace  of  serum  gives  rise  to  the 
rapid  agglutination  and  precipitation  of  1  c.c.  of  mastic  emulsion, 
whereas  such  an  amount  of  the  fresh  physiological  solution  pro- 


348  STUDIES  IN   IMMUNITY. 

duces  no  effect.  If  2  c.c.  of  fresh  blood  of  the  rabbit  is  washed  in 
20  c.c.  of  salt  solution,  the  water  of  washing,  removed  after  cen- 
trifugalizing,  gives  an  immediate  serum  reaction  with  the  mastic. 
If  the  corpuscles  are  washed  a  second  time  with  a  fresh  amount  of 
physiological  solution  and  centrifugalized,  the  supernatant  fluid 
gives  a  like  reaction.  Even  the  third  water  of  washing  gives  a 
positive  result  with  the  mastic  emulsion.  The  fourth  wash  water 
usually  shows  no  evidence  of  the  presence  of  serum. 

It  is  evident  from  these  preliminary  observations  that  enough  pre- 
cipitinogen  is  present  in  the  diluted  serum  which  surrounds  blood 
corpuscles  washed  in  the  ordinary  manner,  to  give  a  precipitate  in 
the  presence  of  immune  serum.  Let  us  now  consider  more  closely 
the  power  of  this  specific  serum  precipitate  to  fix  alexin.  The  work 
of  Gengou*  demonstrated  that  a  serum  active  against  a  foreign  serum 
will,  when  mixed  with  this  causative  albuminoid,  absorb  alexin,  as 
shown  by  the  absence  of  hemolysis  in  sensitized  corpuscles  sub- 
sequently added.  And  the  author  notes  particularly  that  this 
absorption  of  alexin  is  in  proportion  to  the  presence  of  specific 
serum  precipitates;  but  he  did  not  determine  whether  it  is  the 
precipitate  itself  or  some  other  albuminoid  in  solution  that  exer- 
cises this  alexin-fixing  property.  In  the  light  of  the  present  com- 
munication it  is  evident  that  it  is  the  precipitate  itself  that  fixes 
the  alexin. 

The  following  experiment  shows  that  a  specific  serum  precipitate 
will  fix  alexin.  To  furnish  the  precipitinogen  necessary  for  the  for- 
mation of  this  precipitate  I  have  used,  instead  of  dilute  separated 
serum,  the  supernatant  salt  solution  that  had  been  employed  to 
wash  native  blood.  Such  a  serum  dilution  furnishes  the  same 
conditions  as  are  obtained  when  insufficiently  washed  corpuscles 
are  mixed  with  the  immune  serum. 

EXPERIMENT  I. 

To  2  c.c.  of  fresh  ox  blood  is  added  38  c.c.  of  salt  solution  of  0.85 
per  cent;  the  suspension  is  then  centrifugalized  and  the  supernatant 
washing  solution  removed.  The  blood  is  washed  with  another  38 
c.c.  of  the  physiological  solution  and  both  the  washing  fluids  are 
used  for  the  following  experiment: 

*  Gengou,  1.  c. 


THE  FIXATION  OF  ALEXINS.  349 

Tube  1.     First  NaCl  washing  solution  0.2  c.c. 

Serum  rabbit  >  ox,  55  degrees*  0.6  c.c. 

Tube  2.     Second  NaCl  washing  solution  0.2  c.c. 

Serum  rabbit  >  ox,  55  degrees  0.6  c.c. 

Tube  3.     Fresh  NaCl  solution  0.2  c.c. 

Serum  rabbit  >  ox,  55  degrees  0.6  c.c. 

Tube  4.     First  NaCl  washing  solution  0.2  c.c. 

Serum  normal  rabbit,  55  degrees  0.6'  c.c. 
Tubes  are  left  at  room  temperature  2  hours. 
In  tube  1.     Abundant  precipitate. 
In  tube  2.     Trace  of  precipitate. 
In  tubes  3  and  4.     No  precipitate. 

Tube  1  is  then  centrifugalized  and  the  supernatant  fluid  forms 
tube  la,  while  the  precipitate  is  brought  to  the  original  volume 
(0.8  of  a  cubic  centimeter)  with  salt  solution  and  forms  Tube  1.  To 
each  of  the  tubes  1,  la,  2,  3  and  4  is  then  added  fresh  rabbit  serum 
(24  hours),  0.075  of  a  cubic  centimeter,  and  contact  allowed  for  2 
hours  at  room  temperature.  To  each  tube  is  then  added  0.025  of  a 
cubic  centimeter  of  sensitized  rabbit  corpuscles  (S.  rabbit  >  ox, 
55  degrees),  and  the  resultant  hemolysis  is  as  follows: 

Tube  1  (precipitate).     No  hemolysis. 
Tubes  la,  2,  3  and  4.     Hemolysis  complete. 

This  experiment  shows  clearly  that  it  is  the  specific  precipitate 
that  fixes  the  alexin.  In  tube  2  the  hemolysis,  although  finally 
complete,  is  distinctly  delayed  owing  to  partial  absorption  of  the 
alexin  by  the  very  slight  precipitate. 

The  marked  difference  in  dosage  between  the  precipitinogen  and 
the  precipitin  is  indicated  by  the  dilutions  of  ox  serum  represented 
by  the  washing  solutions  of  the  last  experiment.  In  fact,  very 
small  traces  of  the  precipitinogen  suffice  to  give  a  maximum  pre- 
cipitate, provided  sufficient  immune  serum  (precipitin)  is  used. 
A  more  accurate  idea  of  the  relation  of  dosage  and  dilution  between 
the  two  precipitate-forming  sera  than  is  given  incidentally  in  the 
following  experiments  need  not  concern  us  here,  since  we  are  to  deal 
rather  with  the  properties  of  precipitates  than  with  their  formation. 

The  question  may  properly  arise  as  to  whether  the  sensitizing 
activity  of  the  immune  body  for  the  corpuscles  has  been  diminished 
by  the  formation  of  a  specific  precipitate,  and  is  directly  answered 
by  the  following  experiment: 

*  Which  abbreviation  is  used  to  indicate  the  serum  of  a  rabbit  immunized 
against  ox  blood.  Such  serum,  as  indicated,  has  been  heated  to  55°  C  for 
one-half  hour  to  deprive  it  of  alexin. 


350  STUDIES  IN  IMMUNITY. 

EXPERIMENT  II. 
Two  large  tubes  are  prepared. 

Tube  A.     Serum  of  ox,  55  degrees  0.05  c.c. 

Serum  rabbit  >  ox,  55  degrees  2.00  c.c. 

Tube  B.     NaCl  solution,  0.85  per  cent  0.05  c.c. 

Serum  rabbit  >  ox,  55  degrees  2.00  c.c. 

Contact,  2  hours.  Tube  A  gives  a  dense  precipitate;  B,  none. 
Tube  A  is  centrifugalized  and  the  supernatant  fluid  used  for  the 
following  small  tubes: 

Series  A.             Tube  1.  Treated  serum  A  0.2      c.c. 

Tube  2.  Treated  serum  A  0.1      c.c. 

Tube  3.  Treated  serum  A  0.05    c.c. 

Tube  4.  Treated  serum  A  0.025  c.c. 

Series  B.             Tube  5.  Treated  serum  B  0.2      c.c. 

Tube  6.  Treated  serum  B  0.1      c.c. 

Tube  7.  Treated  serum  B  0.05    c.c. 

Tube  8.  Treated  serum  B  0.025  c.c. 

Each  tube  is  brought  to  the  same  volume  (0.2  of  a  cubic  centi- 
meter) with  salt  solution,  and  then  to  each  tube  is  added 

Washed  ox  corpuscles  0.05  c.c.  (washed  four  times) 

Alexin  of  rabbit  0.05  c.c. 

Resultant  hemolysis  is  as  follows : 

Series  A.  Series  B. 

Tube  1.   Complete  Tube  5.   Complete 

Tube  2.   Nearly  complete  Tube  6.   Nearly  complete 

Tube  3.   Marked  Tube  7.    Marked 

Tube  4.    Marked  Tube  8.    Marked 

As  is  evident  from  this  experiment,  the  hemolytic  immune  body 
is  not  affected  by  the  formation  of  the  specific  precipitate. 

The  alexin-fixing  power  of  the  precipitate  is  not  specific  as 
regards  alexin — that  is,  it  is  able  to  absorb  alexins  other  than  those 
of  the  species  furnishing  the  precipitin.  The  fixation  of  guinea- 
pig  alexin,  for  example,  is  shown  by: 

EXPERIMENT  III. 
Two  tubes  are  prepared: 

Tube  1.  Serum  >  ox,  55  degrees  0.025  c.c.  (0.1  c.c.  of  a  dilution  of  1  to  4) 

Serum  rabbit  >  ox,  55  degrees  2.00    c.c. 
Tube  2.  NaCl  solution  0.1       c.c. 

Serum  rabbit  >  ox,  55  degrees  2.00    c.c. 


THE  FIXATION   OF   ALEXINS.  351 

To  each  tube  is  added  alexin  of  the  guinea-pig  one-thirtieth  of  a 
cubic  centimeter,  and  contact  allowed  for  one-half  hour.  In  tube  1 
a  considerable  precipitate  is  formed;  in  tube  2,  none.  Then  to 
each  tube  is  added  0.05  of  a  cubic  centimeter  of  ox  corpuscles 
sensitized  with  S.  rabbit  >  ox,  55  degrees  (1J  hemolytic  doses). 

RESULTANT  HEMOLYSIS. 

Tube  1.     No  hemolysis. 
Tube  2.     Hemolysis  complete. 

Which  shows  that  the  precipitate  has  fixed  the  guinea-pig  alexin. 

II. 

That  a  disregard  of  this  fixation  of  alexin  by  specific  precipitates 
has  led  to  many  erroneous  impressions  of  the  mechanism  of  hemoly- 
sis will  undoubtedly  prove  true,  but  I  wish  to  commit  myself  only 
on  such  phases  of  the  question  as  I  have  been  able  to  submit  to 
experimental  study. 

Recently  Pfeiffer  and  Friedberger*  have  given  the  resume  of  a 
study  of  the  antibacteriolytic,  or  "  antagonistic,"  substances  which 
are  said  to  occur  in  normal  sera.  These  authors  have  found  that 
certain  normal  sera,  which  in  themselves  possess  no  antilytic  proper- 
ties, acquire  distinct  antibacteriolytic  power  when  previously  put 
in  contact  with  the  bacteria  on  which  they  are  destined  subsequently 
to  act.  For  example,  normal  rabbit  serum  treated  with  typhoid 
bacilli  has  the  power  to  prevent  in  vivo  the  destruction  of  sensitized 
typhoid  organisms;  untreated  serum  has  no  such  power,  nor  does 
the  serum  treated  with  typhoid  bacilli  show  any  antilytic  effect 
for  cholera  vibrios  sensitized  with  anticholera  serum.  A  further 
consideration  of  these  most  interesting  observations  concerning 
bacteria  need  not  concern  us  here,  but  an  analogous  series  of  facts 
in  hemolysis,  which  was  soon  published  by  Sachs, f  and  the  con- 
clusions of  this  author  must  be  regarded  more  in  detail.  Normal 
rabbit  serum  heated  to  55  degrees  when  treated  with  equal  parts 
of  sedimented  red  blood  corpuscles  of  the  sheep  or  of  the  pig  inhibits 
the  action  of  guinea-pig  alexin  on  the  properly  sensitized  corpuscles 
of  the  blood  in  question.  Normal  untreated  serum  has  no  such 

*  Pfeiffer  und  Friedberger,  Deutsche  med.  Wochenschr. ,  1905,  No.  1,  p.  6. 
t  Sachs,  Deutsche,  med.  Wochenschr.,  1905,  No.  18,  p.  705. 


352  STUDIES  IN  IMMUNITY. 

antihemolytic  property,  and  the  treated  serum  itself  acts  only  to 
protect  the  species  of  corpuscles  with  which  this  serum  has  been 
digested.  The  explanation  which  Sachs  offers  for  the  facts  up  to 
the  present  point  is  as  follows.  Normal  rabbit  serum  contains 
a  series  of  normal  hemolytic  amboceptors,  of  which  some  are  specific 
for  sheep  corpuscles.  When  treated  with  sheep  corpuscles  rabbit 
serum  loses  its  sheep  amboceptors,  but  the  remaining  amboceptors 
(active  against  other  corpuscles),  although  unattached  to  their 
specific  cells,  have  a  greater  affinity  for  complements  than  do  the 
sheep  corpuscles  sensitized  with  serum  rabbit  >  ox,  added  as  a 
test  for  alexin;  and  hemolysis  of  these  test  corpuscles  does  not  take 
place.* 

If  we  repeat  in  detail  Sachs'  first  experiment,  together  with  a 
control  suggested  by  the  facts  I  have  already  adduced,  it  is  evident 
that  his  explanation  of  this  interesting  phenomenon  is  certainly 
incorrect.  I  have  worked  with  sheep  blood  only  and  have  em- 
ployed the  specific  serum  used  by  Sachs,  that  is,  the  serum  of  a 
rabbit  immunized  against  ox  blood  (which,  of  course,  readily  destroys 
ox  red  blood  corpuscles,  but  also  works  satisfactorily  against  the  red 
cells  of  the  sheep). 

EXPERIMENT  IV. 
Three  tubes  are  prepared  as  follows: 

Tube  A.     Sheep  corpuscles  (the  sediment  of  blood  washed  once 

in  15  volumes  of  NaCl  solution  of  0.85  per  cent  1.5  c.c. 

Normal  rabbit  serum,  55  degrees  1.5  c.c. 

Tube  B.     Normal  rabbit  serum,  55  degrees  1.5  c.c. 
Tube  C.     Sheep  corpuscles  (the  sediment  of  blood  washed  five 

successive  times  with  fresh  volumes  of  NaCl)  1.5  c.c.* 

Normal  rabbit  serum,  55  degrees  1.5  c.c. 

Of  these  tubes,  A  and  B  correspond  exactly  in  dosage  to  those 
given  by  the  German  author.  Just  how  completely  he  washed 

*  As  is  usual  with  the  Ehrlich  school,  an  hypothesis  was  invented  in  harmony 
with  the  lateral-chain  theory,  to  explain  the  Neisser-Wechsberg  phenomenon; 
and  it  is  this  hypothesis  and  not  fundamental  experimental  facts  which  is  used 
as  a  foundation  for  further  hypotheses.  It  has  never  been  proved  that  an  alexin 
can  unite  with  an  immune  body  unless  the  latter  has  formed  a  complex  with  the 
cell  or  substance,  the  injection  of  which  has  given  rise  to  the  specific  serum.  The 
Neisser-Wechsberg  phenomenon,  which  has  been  accepted  by  the  Ehrlich  school 
as  proving  this  union,  is,  in  reality,  unquestionably  due  to  another  cause,  as  I 
shall  consider  later. 


THE  FIXATION   OF   ALEXINS.  353 

the  sheep  corpuscles  he  does  not  state,  but  we  may  presume  not 
far  differently  from  the  manner  employed  in  tube  A,  since  the 
result  is  the  same.  Tube  C  differs  from  tube  A  only  in  the  fact 
that  every  trace  of  sheep  serum  has  been  removed  by  the  repeated 
washings.  The  succeeding  steps  follow  exactly  the  conditions 
and  dosage  of  Sachs. 

Tubes  A,  B  and  C  are  left  at  37°  C  for  1  hour.  Tubes  A  and  C 
are  then  centrifugalized  and  the  supernatant  treated  sera  as  well  as 
the  contents  of  tube  B  serve  to  make  the  following  tubes : 

Tube  1.     Treated  serum  A  1.0  c.c. 

Serum  of  guinea-pig  (alexin)  0.1  c.c. 

Tube  2.  Treated  serum  A  0.2  c.c. 

Alexin,  guinea-pig  0.1  c.c. 

Tube  3.  Serum  B  1.0  c.c. 

Alexin  of  guinea-pig  0.1  c.c. 

Tube  4.  Treated  serum  C  1.0  c.c. 

Alexin,  guinea-pig  0.1  c.c. 

Tube  5.  Treated  serum  C  0.2  c.c. 

Alexin,  guinea-pig  0.1  c.c. 

Contact  at  37  degrees  for  one-half  hour.  Then  to  each  tube  is 
added  1  c.c.  of  a  5  per  cent  suspension  of  washed  sheep  corpuscles 
(5  times)  plus  0.4  of  a  cubic  centimeter  of  serum  rabbit  >  ox, 
55  degrees  (about  two  hemolytic  doses).  Resultant  hemolysis  is 
as  follows: 

Tnhp  1     1  Tube  3  1 

£  K    o  }  No  hemolysis  Tube  4  \  Hemolysis  complete 

Tube  5  ) 

That  is,  in  rabbit  serum  treated  with  imperfectly  washed  sheep 
corpuscles,  there  is  a  substance  that  prevents  the  hemolysis  of  test 
corpuscles  added  at  the  end;  this  is  the  Sachs  experiment.  If  the 
corpuscles  are  washed  so  as  to  remove  all  sheep  serum,  there  is  no 
antagonistic  substance  found.  That  there  is  an  alexin-fixing  sub- 
stance present  in  tube  "A"  is  true,  but  it  is  the  precipitate  formed 
at  the  end  by  the  interaction  of  the  immune  serum  and  the  sheep 
precipitinogen  carried  by  the  treated  rabbit  serum,  and  not  the 
treated  serum  itself.  That  no  true  " anticomplement  action" 
exists  in  the  digested  normal  rabbit  serum  itself  in  the  experiment 
of  Sachs  is  easy  of  proof  and  would  have  been  evident  in  the  tubes 
of  this  experimenter  had  he  only  subjected  the  tubes  which  he 
compares,  to  the  same  experimental  conditions.  The  details  of 


354  STUDIES   IN   IMMUNITY. 

his  last  experiments,  which  show  a  grave  experimental  error,  are 
the  following:  The  normal  rabbit  serum  treated  with  insufficiently 
washed  sheep  corpuscles  brought  about  the  inhibition  of  hemolysis 
already  noted,  when,  for  the  sensitizing  of  the  test  corpuscles,  he  used 
S.  rabbit  >  ox,  55  degrees.  In  this  case  the  excess  of  sensitizing 
serum  was  left  with  the  test  corpuscles,  and  of  course  a  precipitate 
was  formed  in  the  last  stage  of  the  experiment,  and  hemolysis 
thereby  inhibited.  No  such  " anticomplement  action"  took  place 
if  he  sensitized  the  corpuscles  both  with  serum  rabbit  >  ox,  55 
degrees,  and  with  heated  normal  rabbit  serum.  In  this  instance 
he  removed  the  excess  of  both  sensitizing  sera,  and  no  precipitate 
was  formed.  And  again  he  notes  that  no  inhibition  of  hemolysis 
occurred  if,  for  sensitizing  the  test  corpuscles,  he  used  normal  rabbit 
serum  alone.  Incidentally,  the  serum  was  removed  in  this  case, 
but  of  course  no  inhibition  would  have  taken  place  anyway,  as  no 
precipitate  was  formed.*  Manifestly,  the  fixation  of  alexin  (inhibi- 
tion of  hemolysis)  occurs  only  in  the  presence  of  a  precipitate 
formed  by  the  interaction  of  an  excess  of  immune  serum  and  the 
precipitinogen  of  the  sheep  serum  carried  from  the  first  incom- 
pletely washed  corpuscles.  The  following  experiment  comprises  a 
complete  refutation  of  Sachs'  hypothesis  and  puts  in  evidence  the 
alexin-fixing  precipitate : 

EXPERIMENT  V. 
The  tubes  are  prepared  as  follows: 

Tube  A.     Sheep  corpuscles  (washed  once)  3  c.c. 

Serum  normal  rabbit,  55  degrees  3  c.c. 

Tube  B.  Sheep  corpuscles  (washed  five  times)  3  c.c. 

Serum  normal  rabbit,  55  degrees  3  c.c. 
Contact,  1  hour  at  37°  C 

Centrifugalization,  and,  from  the  supernatant  treated  sera  A  and 
B,  are  formed  two  tubes: 

Tube  A1.    Treated  serum  "A"  2.5    c.c. 

Alexin  guinea-pig  0.25  c.c. 

TubeB1.     Treated  serum  "  B "  2.5    c.c. 

Alexin  guinea-pig  0.25  c.c. 

*  Sachs,  I.e.;  compare  Tabelle  2,  Kol.  I  and  II;  Tabelle  3,  Kol.  B,  and  Tabelle  2, 
Kol.  I. 


THE  FIXATION   OF  ALEXINS.  .  355 

Contact,  three-quarters  of  an  hour  at  room  temperature,  and 
then  the  following  tubes  are  made: 

Tube  1.     A1  mixture  1.1  c.c. 

Serum  rabbit  >  ox,  55  degrees  0.4  c.c. 

Tube  2.  A1  mixture  1.1  c.c. 

Serum  normal  rabbit,  55  degrees  0.4  c.c. 

Tube  3.  B1  mixture  1.1  c.c. 

Serum  rabbit  >  ox,  55  degrees  0.4  c.c. 

Tube  4.  B1  mixture  1.1  c.c. 

Serum  normal  rabbit,  55  degrees  0.4  c.c. 

After  contact,  a  precipitate  is  seen  in  tube  1  but  none  in  tubes 
2,  3  and  4.  Then  to  each  tube  is  added  0.05  of  a  cubic  centimeter 
of  washed  sheep  corpuscles  sensitized  with  S.  rabbit  >  ox,  55  degrees, 
and  with  the  excess  of  the  immune  serum  removed  by  centrifugaliz- 
ing. 

The  resultant  hemolysis  is  as  follows: 

Tube  1.     No  hemolysis. 

Tubes  2,  3  and  4.     Hemolysis  complete. 

This  experiment  clearly  demonstrates  that  the  so-called  "am- 
boceptor  anticomplement "  action  of  Sachs  is  simply  due  to  specific 
precipitates. 

As  will  suggest  itself,  the  alexin-fixing  action  of  serum  precipitates 
may  readily  be  brought  forward  to  explain  the  Neisser-Wechsberg 
phenomenon  of  " complement  deviation."  This,  it  will  be  remem- 
bered, was  demonstrated  in  the  case  of  bacteria,  where  it  was  found 
that  an  excess  of  immune  serum  prevented  the  complete  destruction 
of  a  given  dose  of  bacteria  by  an  amount  of  alexin  that  destroyed 
perfectly  the  same  amount  of  organisms  if  the  optimal  dose  of 
immune  serum  were  used.  The  authors  reconciled  these  experi- 
ments with  the  Ehrlich  hypothesis  by  supposing  that  the  mass 
action  of  the  excess  of  free  amboceptor  deviated  the  complement. 
But  no  one  has  been  able  to  demonstrate  the  hemolytic  analogue 
of  this  phenomenon.  Morgenroth,*  it  is  true,  by  making  certain 
suppositions  as  regards  the  union  of  "  complements "  with  free 
amboceptors,  and  by  introducing  certain  other  bodies  ("antiam- 
boceptors"),  has  obtained  somewhat  similar  results,  but  his  analogy 
is  far  from  exact.  The  discussion  of  this  subject,  together  with 
*  Morgenroth,  Centralbl.  f.  Bakt.,  etc.,  Bd.  XXXV,  1904,  p.  504. 


356  STUDIES  IN   IMMUNITY. 

definite  experimental  demonstration  that  deviation  of  the  alexin 
may  exist  in  hemolysis  under  conditions  absolutely  identical  to 
those  described  for  bacteriolysis  by  Neisser  and  Wechsberg,  will 
appear  presently  in  the  Pasteur  Annals.*  I  may  note  simply  that 
it  is  indeed  the  conjectured  role  of  precipitates  that  does  cause  this 
alexin  deviation  in  hemolysis. 

CONCLUSIONS. 

1.  As  was  noted  by  Gengou,  the  serum  of  an  animal  of  species 
A,  injected  with  the  blood  serum  of  species  B,  contains  specific 
"substances  sensibilisatrices "  which,  when  the  immune  serum  A 
is  mixed  with  serum  B,  forms  a  complex  which  fixes  alexin.     This 
alexin-fixing  substance  is  the  specific  serum  precipitate  formed  by 
the  interaction  of  the  two  sera. 

2.  Repeated  washings  of  blood  with  relatively  large  amounts  of 
physiological  solution  are  necessary  to  remove  all  traces  of  serum. 
A  very  small  amount  of  this  serum  contains  enough  precipitinogen 
to  form  a  large  precipitate  if  enough  precipitin  (immune  serum) 
be  present. 

3.  The  formation  of  a  serum  precipitate  does  not  affect  the  sen- 
sitizing strength  of  the  hemolytic  immune  body. 

4.  The  so-called  " anticomplements  of  normal  sera"  of  Sachs 
and  probably  also  the  "antagonistic  substances"  of  Pfeiffer  and 
Friedberger  are  simply  specific  serum  precipitates  capable  of  fixing 
alexin. 

5.  A  disregard  of  the  presence  and  the  alexin-fixing  properties 
of  serum  precipitates  has  doubtless  given  rise  to  many  erroneous 
impressions  of  the  mechanism  of  hemolysis. 

*  See  p.  357. 


XVIII.  DEVIATION  OF  THE  ALEXIN  IN  HEMOLYSIS.* 

BY   FREDERICK   P.   GAY. 

Since  the  observations  of  Neisser  and  Wechsbergf  on  the  inhibiting 
influence  on  bacteriolysis  of  too  large  a  dose  of  specific  immune 
serum  (previously  heated  to  55  degrees)  when  the  amount  of  alexin 
is  relatively  small,  many  attempts  have  been  made  to  determine 
the  mechanism  of  this  interesting  phenomenon.  The  interpreta- 
tion that  Neisser  and  Wechsberg  gave  is  well  known;  it  is  wholly 
in  harmony  with  Ehrlich's  theory.  According  to  these  authors,  in 
a  mixture  of  bacteria,  a  relatively  small  dose  of  alexin,  and  a 
relatively  large  dose  of  heated  immune  serum,  or,  in  other  words,  of 
sensitizer  (amboceptor),  not  all  the  alexin  is  utilized  in  destroying 
bacteria.  Not  all  the  large  amount  of  sensitizer  present  can  be 
absorbed  by  the  bacteria;  the  excess  remains  in  the  surrounding 
fluid  and,  owing  to  its  affinity  for  alexin,  takes  up  a  greater  or  less 
amount  of  this  substance  that  consequently  is  of  no  service  in 
producing  bacteriolysis.  The  inhibiting  effect  of  too  much  immune 
serum  would  be  explained  as  a  real  deviation  of  the  complement 
(Komplementablenkung)  by  the  excess  of  sensitizer  not  combined 
with  the  bacteria.  This  hypothesis,  to  be  sure,  has  neither  been 
experimentally  proved  nor  refuted.  It  is  by  no  means  necessary  to 
accept  it,  as  it  is  based  on  a  supposition  that  has  never  been  proved 
experimentally,  namely,  that  the  sensitizing  substance  can  fix 
alexin  even  when  uncombined  with  bacteria.  It  must  be  admitted 
that  certain  defenders  of  the  hypothesis,  notably  Lipstein,f  have 
been  able  to  reply  to  certain  objections  that  have  been  raised  to  it, 
without,  however,  bringing  forward  any  really  definite  proofs  of  its 
correctness.  The  inhibiting  effect  of  an  excess  of  immune  serum 

*  La  deviation  de  1'alexine  dans  1'hemolyse.    Annales  de  Tlnstitut  Pasteur, 

XIX,  1905,  593. 

t  See  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  &  Sons,  p.  120. 
J  Lipstein,  Ehrlich-Bolduan,  Wiley  &  Sons,  p.  132. 

357 


358  STUDIES  IN   IMMUNITY. 

evident  in  bacteriolysis  has  never  been  shown  to  exist  in  hemolysis. 
The  experiments  by  Morgenroth*  demonstrating  such  a  phenomenon 
in  hemolysis  are  evidently  incorrectly  conceived,  as  Bordetf  has 
shown,  and  should  not.be  admitted  as  evidence. 

The  interpretation  we  wish  to  offer  of  this  Neisser  and  Wechsberg 
phenomenon  has  an  evident  relation  to  certain  experimental  results 
that  have  already  been  described.!  These  experiments  dealt  with 
the  alexin-fixing  action  of  the  albuminous  precipitates  obtained  by 
mixing  a  precipitin  serum  with  the  proper  antigen.  The  results 
may  be  summarized  as  follows : 

1.  Gengou  §  showed  that  the  serum  of  animals  of  species  A 
immunized  against  an  albuminous  substance  of  species  B  has  not 
only  a  precipitating  property  for  these  substances,  but  also  endows 
them  with  the  property  of  absorbing  alexin.     These  albuminous  sub- 
stances treated  with  the  specific  immune  serum  act,  in  other  words, 
as  do  blood  corpuscles  or  bacteria  treated  with  suitable  sensitizers. 

2.  We  were  able  to  carry  Gengou's  observations  farther  by  show- 
ing that  the  fixation  of  alexin  in  this  instance  is  brought  about 
exclusively  by  the  precipitated  albuminous  substances  and  not  by 
those  in  solution. 

3.  It  is  worth  noting  that  a  very  weak  dilution  of  precipitinogen 
(precipitable  serum)  in  salt  solution  will  give  a  relatively  abundant 
precipitate  with  a  suitable  dose  of  precipitin.    To  avoid  the  occur- 
rence of  a  serum  precipitate  in  hemolytic  experiments,  it  is  indis- 
pensable to  wash  the  corpuscles  employed  several  times  with  salt 
solution  to  remove  the  accompanying  serum  entirely.    This  serum 
if  present  will  form  a  precipitate  with  the  immune  serum  used  to 
sensitize  the  blood  corpuscles  in  such  an  experiment. 

4.  This  precaution  in  washing  has  apparently  not  been  taken 
by  many  investigators ;  as  a  result,  in  their  mixtures  of  incompletely 
washed  blood  (that  is,  blood  containing  traces  of  serum)  and  hemo- 
lytic substances  (sensitizer  and  alexin)  a  precipitate  was  formed 
that  could  independently  fix  a  greater  or  less  amount  of  the  alexin 
employed.    This  fixed  alexin  would,  of  course,  not  affect  the  cor- 
puscles, and  consequently  a  true  deviation  of  alexin  would  have 
occurred  in  a  hemolytic  experiment. 

*  Centralblatt  f.  Bakt.,  XXV,  1904,  501.  f  See  p.  299. 

%  See  p.  346.  $  See  p.  241. 


DEVIATION   OF  THE  ALEXIN   IN  HEMOLYSIS.  359 

5.  This  last  paragraph  is  applicable  to  certain  recent  researches 
of  Sachs*  on  the  so-called  " anticomplement "  power  of  normal 
sera.  For  the  discussion  of  these  experiments  we  may  refer  to  the 
previous  article.  Pfeiffer  and  Fried berger  have  also  observed 
certain  analogous  facts  with  bacteria,  and  it  would  seem  as  if  their 
work  might  be  explained  in  a  similar  manner. 

We  shall  now  consider  more  attentively  the  experimental  results 
to  which  reference  has  been  made  in  paragraphs  2  and  3  above. 
We  have  seen,  first,  that  the  albuminous  substances  precipitated  by 
a  specific  serum  are  in  reality  sensitized,  or,  in  other  words,  able  to 
absorb  alexin,  and,  secondly,  that  small  amounts  of  precipitinogen 
suffice  to  form  an  abundant  precipitate  with  the  precipitin  serum. 
We  must  further  emphasize  that  a  relatively  large  amount  of  this 
precipitin  serum  is  necessary  to  cause  a  precipitum  sufficient  to 
fix  alexin  well.  The  following  experiment  indicates  the  relation 
between  the  two  sera,  the  precipitum  obtained,  and  the  degree  of 
alexin  absorption.  As  precipitinogen,  ox  serum  is  used,  and,  as 
precipitin,  the  serum  of  a  rabbit  immunized  against  ox  blood  and 
heated  to  55  degrees.! 

Tube  1.     Ox  serum,  55  degrees  0.025  c.c. 

Serum  rabbit  >  ox,  55  degrees  0.1      c.c. 

NaCl  solution  (0.85  per  cent)  1.9  c.c. 

Tube  2.  Ox  serum  0.025  c.c. 

Serum  rabbit  >  ox,  55  degrees  0.6      c.c. 

NaCl  solution  1.4  c.c. 

Tube  3.  Ox  serum  0.025  c.c. 

Serum  rabbit  >  ox,  55  degrees  2.0      c.c. 

These  three  tubes  contain  the  same  total  volume  and  the  same 
amount  of  ox  serum.  They  differ  in  their  respective  amounts  of 
precipitin  serum,  which  in  the  small  doses  is  replaced  by  salt  solu- 
tion, so  that  the  total  volumes  are  the  same.  Three  control  tubes 
(4,  5  and  6)  are  also  prepared  corresponding  to  tubes  1,  2  and  3 
respectively,  but  containing  salt  solution  in  place  of  ox  serum. 
After  one-half  hour  at  37  degrees  there  is  no  evident  precipitate 
in  tubes  1,  4,  5  and  6;  there  is  a  distinct  though  slight  precipitate 
in  tube  2;  and  in  tube  3  there  is  a  voluminous  precipitate. 

*  Deutsche  med.  Woch.,  1905,  No.  18,  705. 

t  This  serum,  for  the  sake  of  abbreviation,  is  referred  to  as  "  Sorum  rabbit  >  ox.  '* 


360  STUDIES  IN  IMMUNITY. 

To  each  tube  there  is  then  added  one-thirtieth  of  a  cubic  centi- 
meter of  normal  rabbit  alexin  (serum  from  blood  obtained  the  day 
before).  Fifteen  minutes  later  0.3  of  a  cubic  centimeter  of  sen- 
sitized ox  blood  is  added  to  each  tube.  This  sensitized  blood  is  a 
mixture  of  one  part  of  carefully  washed  ox  blood  *  to  five  parts  of 
heated  rabbit  >  ox  serum. 

There  is  complete  hemolysis  in  tubes  1,  4,  5  and  6;  partial  hemoly- 
sis  in  tube  2;  and  no  hemolysis  in  tube  3.  The  alexin,  therefore,  has 
remained  free  in  tubes  1,  4,  5  and  6  containing  no  precipitate,  has 
been  partially  absorbed  in  tube  2  containing  a  moderate  precipitate, 
and  has  been  completely  absorbed  in  tube  3  that  contains  the  largest 
precipitate.  In  other  words,  the  greater  the  amount  of  precipitate 
the  greater  the  alexin  absorption  and  corresponding  absence  of 
hemolysis. 

As  may  be  imagined,  this  experiment  may  be  performed  some- 
what differently.  A  mixture  containing  the  albuminous  precipitum 
and  the  sensitized  corpuscles  may  be  made  and  the  alexin  sub- 
sequently added.  Under  these  conditions  we  should  expect  that 
both  sensitized  elements  that  are  avid  of  alexin,  namely,  the  pre- 
cipitate and  the  corpuscles,  would  struggle  to  obtain  the  alexin, 
and  hemolysis  would  be  more  or  less  inhibited  or  even  entirely  pre- 
vented if  the  dose  of  alexin  were  not  too  large.  To  produce  these 
conditions  we  may  place  in  tube  A  small  amounts  of  well-washed 
ox  blood  (0.05  of  a  cubic  centimeter)  and  of  ox  serum  (0.025  of  a 
cubic  centimeter),  and  then  add  a  very  large  dose  of  heated  rabbit 
>  ox  serum  (2.5  c.c.).  A  few  seconds  later  let  us  add  rabbit  alexin 
(one-thirtieth  of  a  cubic  centimeter).  There  is  no  hemolysis,  as  the 
precipitum  has  absorbed  all  the  alexin.  In  a  control  tube  B  pre- 
pared at  the  same  time,  and  containing  the  same  components  except 
the  ox  serum,  hemolysis  is  complete,  as  no  precipitate  forms.  All 
that  is  necessary  to  produce  hemolysis  in  tube  A  is  to  add  a  small 
additional  dose  of  alexin. 

We  have  already  noted  that  considerable  immune  serum  is 
necessary  to  obtain  an  abundant  precipitate  with  ox  serum.  Con- 
sequently, if  a  dose  of  rabbit  >  ox  serum  barely  sufficient  to  sensitize 

*  This  blood  had  been  carefully  washed  in  normal  salt  solution  and  then 
restored  to  its  original  volume;  it  corresponds,  then,  to  blood  in  which  the  original 
serum  is  replaced  by  salt  solution. 


DEVIATION   OF  THE  ALEXIN  IN  HEMOLYSIS. 


361 


the  corpuscles,  but  too  little  to  produce  an  abundant  precipitate,  had 
been  put  in  tube  A  we  should  expect  that  hemolysis  would  have 
taken  place.  This  may  be  experimentally  verified.  In  a  tube 
containing  0.025  of  a  cubic  centimeter  of  ox  serum,  0.05  of  a  cubic 
centimeter  of  corpuscles  and  only  0.2  of  a  cubic  centimeter  of 
immune  serum,  hemolysis  occurs  as  well  as  in  a  control  that  con- 
tains no  ox  serum. 

In  such  experiments,  however,  it  is  evident  that  if  the  amount  of 
rabbit  >  ox  serum  is  too  much  diminished  no  hemolysis  will  occur 
owing  to  the  fact  that  the  corpuscles  are  not  sufficiently  sensitized. 

In  the  following  tables  are  given  the  results  of  experiments  in 
which  the  amounts  of  rabbit  >  ox  serum  vary.  In  table  A  the 
mixtures  contain  ox  serum;  in  B  there  is  no  ox  serum,  but  salt 
solution  in  its  place;  the  doses  of  ox  corpuscles  and  rabbit  alexin 
are  constant. 

TABLE  A. 


Tube. 

Ox  serum,  55°. 

Ox  corpuscles. 

Rabbit  >  ox 
serum,  55°. 

NaCl 
sol. 

Rabbit  alexin. 

Hemolysis. 

1 

0.025  c.c. 

0.05  c.c. 

0.05  c.c. 

2.45c. 

0.033+c.c. 

incomplete 

2 

0.025  c.c. 

0.05  c  c. 

0.1    c.c. 

2.4   c. 

0.033+c.c. 

complete 

3 

0.025  c.c. 

0.05  c.c. 

0.2    c.c. 

2.3   c. 

0.033  +  c.c. 

complete 

4 

0.025c  c. 

0.05  c.c. 

0.3    c.c. 

2.2    c. 

0.033+c.c. 

complete 

5 

0.025  c.c. 

0.05  c.c. 

0.6    c.c. 

1.9    c. 

0.033+c.c. 

incomplete 

6 

0.025  c.c. 

0.05  c  c. 

1.0    c.c. 

1.5    c. 

0.033+c.c. 

slight 

7 

0.025  c.c. 

0.05  c.c. 

2.0    c.c. 

0.5    c. 

0.033+c.c. 

none 

8 

0.025  c.c. 

0.05  c.c 

2.5    c.c. 

0.0    c. 

0.033+c.c. 

none 

TABLE  B 


Tube. 

NaCI. 

Ox  cor- 
puscles. 

Rabbit  >  ox 
serum,  55°. 

NaCl  sol. 

Rabbit  alexin. 

Hemolysis. 

1 

0.025  c.c. 

0.05  c.c 

0.05  c.c. 

2.45  c  c. 

0.033+C.c. 

incomplete 

2 

0.025  c.c. 

0.05  c.c. 

0.1    c.c. 

2.4    c  c. 

0.033  +  c  c 

complete 

3 

0.025  c.c. 

0.05  c.c. 

0.2    c.c. 

2.3    c.c. 

0.033  +  c  c 

complete 

4 

0.025  c.c. 

0.05  c.c. 

0.3    c.c. 

2.2    cc. 

0.033  +  c  c. 

complete 

5 

0.025  c.c. 

0.05  c.c. 

0.6    c.c. 

1.9    c  c. 

0.033+c.c. 

complete 

6 

0.025C  c. 

0.05  c.c. 

1.0    c.c. 

1.5    cc. 

0.033+c  c. 

complete 

7 

0.025  c.c. 

0.05  c.c. 

2.0    c.c. 

0.5    c  c. 

0  033+c  c. 

complete 

8 

0.025  c.c. 

0.05  c.c. 

2.5    c.c. 

0.0    c  c. 

0.033+c.c. 

complete 

362  STUDIES  IN  IMMUNITY. 

These  experiments  show  a  Neisser-Wechsberg  phenomenon  in 
hemolysis.  The  result  may  be  described  by  saying  that  blood 
(i.e., serum  plus  corpuscles)  is  not  hemolyzed  by  a  small  dose  of  alexin 
unless  the  .amount  of  sensitizer  is  suitably  small.  Too  much  sen- 
sitizer  protects  the  corpuscles  by  deviating  the  alexin.  This 
deviation  (Komplemenablenkung) ,  however,  is  not  to  be  explained 
as  Neisser  and  Wechsberg  have  done.  It  is  not  due  to  alexin 
absorption  by  means  of  a  certain  amount  of  uncombined  sensi- 
tizer remaining  free  in  the  fluid.  The  fixation  of  alexin  is  brought 
about  by  a  sensitized  precipitum  that  competes  successfully  with 
the  corpuscles  in  absorbing  this  active  substance. 

It  is  particularly  to  be  noted  that  no  Neisser  and  Wechsberg 
phenomenon  occurs  if  the  corpuscles  employed  have  been  washed 
free  of  the  serum  present  in  the  primitive  blood. 

Will  this  interpretation  of  alexin  fixation  in  hemolysis  account 
for  the  Neisser-Wechsberg  phenomenon  in  bacteriolysis?  We 
expect. to  take  up  this  question  at  a  later  time.  It  may  be  noted 
here  that  a  culture  or  emulsion  of  bacteria  contains  elements  that 
correspond  rather  closely  to  those  present  in  blood.  The  bacteria 
correspond,  of  course,  to  the  corpuscles,  the  bacterial  precipitino- 
gens,  that,  as  Kraus  has  shown,  rorm  precipitates  with  the  specific 
antiserum,  moreover  correspond  to  the  albuminous  substances  in 
serum. 


XIX.      ON    THE    RELATIONS    OF    SENSITIZERS    TO 

ALEXIN* 

BY   DRS.    J.   BORDET   AND   F.   P.    GAY. 

Those  who  have  made  a  study  of  hemolysis  hold  very  divergent 
opinions  as  to  the  relations  between  the  susceptible  corpuscle  and 
the  substances  that  affect  it,  namely,  the  sensitizer  (amboceptor) 
and  the  alexin  (complement).  It  is  well  known  that  the  blood 
corpuscles  fix  the  sensitizer  (Ehrlich  and  Morgenroth),  and  that 
corpuscles  so  modified  have  the  new  property  of  energetically 
absorbing  all  the  alexin  from  the  surrounding  fluid.  (Bordet.) 
It  is  evident,  then,  from  these  facts  that  the  sensitizer  acts  as  an 
intermediary  agent  in  bringing  about  the  union  between  the  sen- 
sitive cell  and  the  toxic  substance  or  alexin. 

It  is  perfectly  evident  that  there  is  some  not  well  understood 
substance  in  the  red  blood  cell  which  unites  and  forms  a  complex 
with  the  sensitizer.  This  much  and  no  more  has  been  experimen- 
tally demonstrated ;  the  intimate  nature  of  the  reaction  is  unknown. 

It  is  scarcely  profitable  to  explain  this  simple  fact  in  any  elaborate 
fashion.  To  say  that  the  corpuscle  receives  and  holds  the  sen- 
sitizer by  means  of  a  "receptor,"  or  that  the  sensitizer  combines  with 
such  a  receptor  because  it  has  a  combining  cytophilic  group,  is  to 
pretend  to  a  knowledge  not  yet  obtained.  We  may  content  our- 
selves by  saying  that  a  complex  is  formed. 

But  how  does  this  complex  (sensitizer-corpuscle)  fix  alexin? 
To  which  constituent  of  the  complex  is  the  affinity  for  this  sub- 
stance due?  It  is  certainly  not  the  corpuscle  itself,  for  we  find  that 
normal  unsensitized  corpuscles  do  not  take  up  alexin.  Is  it,  then, 
the  sensitizer,  or  is  it  the  combination  of  the  two,  that  shows  an 
avidity  for  alexin  that  neither  one  of  its  constituents  alone  possesses? 

Both  hypotheses  have  been  suggested.    The  first  one,  namely, 

*  Sur  les  relations  des  sensibilisatrices  avec  1'alexine.  Annales  de  1'Institut 
Pasteur,  XX,  1906,  467. 

363 


364  STUDIES  IN   IMMUNITY. 

that  the  sensitizer  combines  with  the  alexin,  has  been  suggested  by 
Ehrlich  and  Morgenroth.  According  to  these  authors  the  sensi- 
tizer molecule  has,  in  addition  to  the  atom  group  that  binds  it  to 
the  cell  receptor  (cytophilic  group),  a  second  distinct  group  (com- 
plementophilic)  that  unites  with  the  alexin.  This  explanation,  which 
regards  the  sensitizer  as  a  bond  of  union  between  corpuscles  and 
alexin,  suggests  two  corollaries.* 

In  the  first  place  the  corpuscle  has  no  direct  participation  in 
alexin  absorption,  but  simply  takes  hold  of  the  sensitizer  and  then 
ceases  to  function.  It  is,  then,  the  sensitizer  that  comes  into  play 
in  alexin  absorption  by  means  of  its  own  affinities.  And  further, 
on  Ehrlich  and  Morgenroth 's  thesis,  alexin  absorption  is  a  purely 
chemical  reaction  due  to  affinities  between  atoms  or  groups  of 
atoms.  The  complex  receptor-sensitizer-alexin  may  be  regarded 
as  a  single  large  molecule,  the  nucleus  of  which  is  the  sensitizer  and 
the  side  chains  of  which  are  the  receptor  and  the  alexin. 

The  alexin,  then,  would  unite  with  a  new  and  definite  chemical 
complex,  and  its  absorption  is  not  comparable  with  phenomena  of 
adhesion  (the  fixation  of  a  toxin  by  a  precipitate,  for  example)  nor 
with  the  various  dyeing  phenomena,  in  which  cases  the  molecules 
of  the  substance  to  be  stained  attract  the  molecules  of  the  dye 
without  intervention  of  atomic  affinities.  Nor  is  it  comparable 
to  the  common  facts  observed  in  the  precipitation,  agglutination 
and  coagulation  of  colloidal  substances,  nor,  in  short,  to  the  many 
phenomena  due  to  molecular  adhesion. 

According  to  the  second  explanation,  proposed  several  years  ago 
by  one  of  the  authors  of  the  present  article,  the  sensitizer  does  not 
of  itself  combine  with  the  alexin.  It  unites  with  the  corpuscles  and 
forms  a  complex  which  has  the  new  property  of  uniting  with  the 
alexin  and  of  removing  it  from  the  surrounding  fluid ;  in  other  terms, 
neither  the  proper  substance  of  the  corpuscle  nor  the  sensitizer  by 
itself  has  any  perceptible  affinity  for  the  alexin.  Such  an  affinity 
becomes  evident  only  when  the  proper  substance  in  the  corpuscle 
has  become  modified  (sensitized)  as  a  result  of  union  with  the 
sensitizer  and  so  changed  into  an  alexin-attracting  complex. 

According  to  this  hypothesis,  there  is  no  question  of  a  comple- 

*  In  accordance  with  this  conception  Ehrlich  and  Morgenroth  have  given  the 
name  of  amboceptor  to  the  sensitizer. 


RELATIONS  OF  SENSITIZERS  TO  ALEXIN.  365 

mentophilic  group  in  the  sensitizer  fitted  to  take  hold  of  alexin  with- 
out intervention  of  the  corpuscle;  the  corpuscle  itself  takes  part 
in  the  fixation  inasmuch  as  it  is  one  of  the  constituent  members 
of  the  absorbing  complex.* 

It  is  to  be  noted  that  this  second  point  of  view,  in  accordance 
with  which  the  sensitizer  has  no  complementophilic  group,  is 
much  less  compatible  with  the  idea  that  alexin  fixation  is  a  true 
chemical  reaction,  implying  the  formation  of  a  new  well-defined 
compound. 

Unless  we  suppose  that  the  union  of  the  sensitizer  with  the  cor- 
puscle receptor  is  more  than  a  simple  attachment,  and  unless  we 
admit  that  this  union  modifies  the  affected  molecules  very  pro- 
foundly by  causing  a  new  distribution  of  their  atoms,  it  is  difficult 
to  see  how  this  combination  can  give  rise  to  atom  groups  avid  of 
alexin  when  no  trace  of  them  is  present  in  either  of  the  two  bodies 
that  take  part  in  the  reaction. 

This  latter  conception,  however,  harmonizes  with  the  idea  that 
the  alexin  is  taken  out  and  absorbed  by  a  process  of  molecular 
adhesion  rather  than  by  a  true  chemical  reaction. 

We  have  simply  to  consider  the  substance  of  the  corpuscle  uniting 
with  the  sensitizer  as  so  modified  in  its  properties  of  adhesion  as 
to  form  a  complex  capable  of  sticking  to  alexin,  as  calcium  fluoride 
and  other  inert  chemical  precipitates  in  fine  colloidal  suspension 
remove  the  fibrinogen  from  plasma. 

Do  we  not  find  analogous  if  not  identical  instances  in  the  change 

*  Ehrlich  and  Morgenroth,  to  be  sure,  have  subsequently  modified  their  original 
theory,  at  least  as  regards  certain  sensitizers  or  alexins  and  certain  corpuscles. 
They  admit  that  in  certain  instances  the  sensitizer  shows  no  affinity  for  alexin 
until  it  is  combined  with  the  cell.  It  is  naturally  difficult  to  discuss  or  appraise 
the  value  of  a  theory  that  changes  so  markedly  from  year  to  year. 

To  state  that  the  sensitizer  combines  with  the  alexin  only  after  union  with  the 
corpuscles  is  practically  to  adopt  Bordet's  conception,  according  to  which  neither 
one  of  the  constituents  alone  is  able  to  fix  alexin.  Such  a  statement,  moreover, 
renounces  the  theory  that  was  so  definitely  stated  at  first,  namely,  that  the  alexin 
unites  with  the  sensitizer  even  when  no  corpuscles  are  present. 

If  the  intervention  of  the  corpuscles  is  admittedly  necessary,  the  two  theories 
really  differ  only  in  logic  subtleties. 

It  is  only  fair  to  add  that  although  Ehrlich  and  Morgenroth  admit  that  the 
sensitizer  in  certain  instances  can  combine  only  after  cell  union,  they  state  that  in 
other  cases  the  opposite  condition  occurs,  namely,  that  the  union  of  the  alexin 
with  the  sensitizer  increases  the  affinity  of  the  latter  for  the  corpuscle. 


366  STUDIES  IN   IMMUNITY 

of  molecular  adhesion  that  is  the  essential  cause  of  the  agglutination 
of  bacteria?  Is  it  not  a  change  in  molecular  adhesion  that  makes 
bacteria  treated  by  an  agglutinin  flock  out  when  sodium  chloride 
is  added,  although  normal  bacteria  do  not? 

From  this  viewpoint  alexin  fixation  assumes  a  more  real  and 
general  interest  than  was  at  first  apparent.  In  endeavoring  to 
understand  the  reactions  that  take  place  in  the  body  by  comparison 
with  simpler  and  better-known  facts,  is  it  necessary  to  look  for 
analogies  in  pure  chemistry  alone,  as  do  Ehrlich  and  Morgenroth? 
Such  phenomena,  as  we  know,  follow  laws  of  definite  proportions, 
and  give  rise  to  compounds  of  unvarying  constitutions  described 
by  a  formula.  But  may  we  not  also  cite  analogies  among  the 
phenomena  of  molecular  adhesion,  flocculation,  coagulation,  emul- 
sion, dyeing,  stickiness  and  the  like? 

When  we  try  to  prove  the  principal  proposition  of  Ehrlich  and 
Morgenroth  experimentally,  namely,  that  the  sensitizer  can  com- 
bine with  the  alexin  without  the  presence  of  corpuscles,  negative 
results  are  obtained ;  the  alexin  remains  quite  free,  as  was  demon- 
strated in  earlier  experiments,  to  which  the  reader  is  referred.* 

In  endeavoring  to  prove  their  assertion  Ehrlich  and  Sachsf  have 
laid  much  stress  on  the  supposed  complement  deviation  evidenced 
by  the  well-known  experiments  of  Neisser  and  Wechsberg  on  the 
antibacteriolytic  effect  of  an  excess  of  sensitizer  in  presence  of  a 
relatively  small  dose  of  alexin. 

This  complement  deviation  is  due,  according  to  the  authors  in 
question,  to  the  fact  that  the  excess  of  sensitizer  which  is  refused  by 
the  saturated  bacteria  remains  free  in  the  fluid  and  takes  up  on 
its  own  account  part  of  the  alexin  and  so  prevents  it  from  destroy- 
ing bacteria;  this  supposition  was  never  proved  experimentally. 
Several  authors  have  recently  questioned  Neisser  and  Wechsberg's 
explanation  of  their  phenomenon  and  have  offered  new  explanations 
which  have  no  part  with  the  thesis  defended  by  the  Ehrlich  school. 
(Gay,J  Moreschi,§  Buxton.||) 

*  Bordet,  The  cytolytic  sera,  etc.,  p.  228. 

t  Ehrlich  and  Sachs,  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  & 
Sons,  p.  217. 

t  Gay,  see  p.  357. 

$  Moreschi,  Berliner  klinische  Wochcnschrift,  1906,  p.  100. 

II  Buxton,  Journal  Med.  Research,  XII,  1(J05,  431. 


RELATIONS   OF  SENSITIZERS  TO  ALEXIN.  367 

In  their  studies  on  snake  venoms  Kyes  and  Sachs  *  found  an 
apparent  confirmation  of  this  explanation  of  complement  devia- 
tion, but  Hideyo  Noguchi  f  later  showed  that  their  phenomena  were 
to  be  explained  quite  differently. 

There  is,  then,  no  valid  reason  for  conceiving,  as  Ehrlich  and 
Morgenroth  do,  that,  when  an  alexin  is  not  able  to  destroy  a  given 
corpuscle  sensitized  with  a  certain  sensitizer,  the  failure  is  due  to 
the  inability  of  the  alexin  to  combine  with  the  sensitizer  in  question. 
The  fact  that  an  alexin  does  or  does  not  destroy  in  a  given  case  is 
not  dependent  on  its  adaptability  or  non-adaptability  to  a  sup- 
positions complementophilic  group  of  the  sensitizer  employed. 
The  designations  "passende"  or  "nicht  passende  Komplemente" 
do  not  indicate  any  real  condition. 

And,  indeed,  with  what  alexin  should  a  sensitizer  in  horse  serum 
most  readily  unite  logically?  It  is  evident  that  it  should  combine 
with  an  alexin  from  the  same  animal  species  —  the  horse.  But  as  a 
matter  of  fact,  although  horse  serum  contains  a  sensitizer  that 
hemolyzes  guinea-pig  corpuscles  in  conjunction  with  guinea-pig 
alexin,  no  such  result  occurs  with  horse  alexin. 

Are  we  to  suppose  that  this  sensitizer  unites  with  the  first  alexin 
better  than  with  the  second?  Or  is  it  not  more  reasonable  to  con- 
clude that  both  alexins  are  absorbed  by  the  sensitized  corpuscles, 
but  that  the  horse  alexin  is  simply  less  toxic  and  less  liable  to  cause 
hemolysis?  J 

As  another  example  we  may  note  that  normal  rabbit  serum 
contains  a  sensitizer  for  goat  corpuscles.  This  sensitizer,  however, 
is  much  more  effective  with  guinea-pig  alexin  (which  alone  does 
not  hemolyze,  as  the  serum  of  the  guinea-pig  contains  no  sensitizer) 
than  with  rabbit  alexin.  § 

*  Kyes  and  Sachs,  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  & 
Sons,  p.  443. 

t  Noguchi,  Jour,  of  Exp.  Medicine,  VII,  1905. 

t  In  the  same  way  we  shall  later  show  that  although  horse  alexin  is  well 
absorbed  by  sensitized  ox  corpuscles  it  fails  to  hemolyze  them.  Analogous 
instances  have  already  been  noted  by  Muir  (Proceedings  of  the  Royal  Society, 
Vol.  74,  1904,  305)  and  by  Gay  (this  volume,  p.  336). 

$  EXPERIMENT.  One  cubic  centimeter  of  a  10  per  cent  suspension  of  goat 
blood  in  salt  solution  is  placed  in  each  of  four  tubes.  To  tube  "a"  is  added  0.4 
of  a  cubic  centimeter  of  fresh  rabbit  serum,  to  tube  " b"  0.2  of  a  cubic  centimeter 
of  fresh  guinea-pig  serum,  to  tube  "c"  0.2  of  a  cubic  centimeter  of  rabbit  serum 


368  STUDIES  IN   IMMUNITY 

The  important  factor  that  Ehrlich  and  Morgenroth  neglect  is 
that  each  alexin  has  a  definite  toxic  property  of  its  own  for  a  given 
corpuscle.  It  is  quite  evident  that  the  hemolytic  or  bacteriolytic 
power  of  serum  will  vary  in  different  animal  species  as  their  alexins 
differ  in  intensity. 

There  is,  however,  one  experiment  reported  by  Ehrlich  and 
Sachs*  which,  if  the  interpretation  they  give  to  it  were  correct, 
would  prove  indisputably  that  the  alexin  really  unites  with  the 
sensitizer.  It  would  actually  seem  in  the  experiment  in  question 
as  if  the  sensitizer  does  unite  with  the  corpuscles  subsequent  to  its 
union  with  the  alexin. 

This  alexin-sensitizer  union  would  seem  to  be  so  indispensable 
that,  if  the  alexin  were  destroyed,  a  real  paralysis  of  the  sensitizer 
would  result,  so  that  its  affinity  for  the  corpuscle  would  be  destroyed 
or  at  least  inhibited;  in  other  words,  the  cytophilic  group  would 
seem  to  react  with  the  corpuscle  only  after  the  complementophilic 
group,  uniting  with  the  alexin,  is  satisfied. 

It  remains  to  be  seen,  however,  whether  Ehrlich  and  Sachs  have 
not  entirely  misinterpreted  their  own  experiment.  The  sensitizer 
in  question  is  present  in  inactivated  (56  degrees)  normal  bovine 
serum,  the  corpuscles  affected  are  from  the  guinea-pig,  and  the 
alexin  employed  is  in  the  form  of  fresh  horse  serum. 

We  may  first  of  all  summarize  the  facts  that  Ehrlich  and  Sachs 
have  noted.  Bovine  serum,  inactivated  at  56  degrees,  naturally 
does  not  hemolyze  guinea-pig  corpuscles,  as  its  alexin  has  been 
destroyed.  Fresh  (alexic)  horse  serum  also  has  only  the  slightest 
hemolytic  effect  on  these  cells.  The  heated  ox  serum,  however, 
apparently  sensitizes  the  corpuscles  so  that  they  are  hemolyzed  when 
fresh  horse  serum  is  added.  Strong  hemolysis  (Experiment  I)  is 
evident  when  guinea-pig  corpuscles,  heated  bovine  serum  and  fresh 
horse  serum  are  mixed  together  in  suitable  doses,  t  So  far  there  is 

plus  0.1  of  a  cubic  centimeter  of  guinea-pig  serum,  and  to  tube  "d  "  0.4  of  a  cubic 
centimeter  of  rabbit  serum  plus  0.1  of  a  cubic  centimeter  of  guinea-pig  serum. 
Resultant  hemolysis.  Complete  in  one-half  hour  in  "d";  complete  in  one  hour 
in  "c."  There  is  only  a  trace  of  hemolysis  in  "a"  and  none  in  "b"  in  an  hour. 

*  Ehrlich  and  Sachs,  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  & 
Sons,  p.  209. 

f  For  example,  1  c.c.  of  a  5  per  cent  suspension  of  guinea-pig  blood  plus  0.3  of 
a  cubic  centimeter  of  bovine  serum  (56  degrees)  plus  0.5  of  a  cubic  centimeter  of 
fresh  horse  serum. 


RELATIONS   OF  SENSITIZERS  TO  ALEXIN.        -          369 

nothing  remarkable  in  the  experiment.  But  in  a  second  experiment 
the  corpuscles  are  placed  in  contact  with  the  ox  serum,  and  after 
a  certain  period  the  mixture  is  centrifugalized  and  the  supernatant 
fluid  decanted.  On  the  addition  of  horse  serum  to  .the  sedimented 
corpuscles  no  hemolysis  takes  place.  It  would  seem,  then  (and 
such  is  the  conclusion  of  Ehrlich  and  Sachs),  that  the  corpuscles 
have  failed  to  absorb  the  sensitizer  from  the  ox  serum  in  spite  of 
contact  with  it.  This  conclusion  seems  further  corroborated  by  the 
fact  that  the  supernatant  fluid  that  has  been  used  in  treating  the 
corpuscles  is  just  as  active  as  before  contact.  Fresh  guinea-pig 
corpuscles  when  added  to  this  supernatant  fluid  plus  fresh  horse 
serum  are  readily  hemolyzed.  And,  what  is  more,  the  treated  cor- 
puscles, which  are  not  hemolyzed  by  fresh  horse  serum  alone,  are 
destroyed  as  readily  as  fresh  corpuscles  by  a  mixture  of  heated  ox 
serum  and  horse  alexin. 

Ehrlich  and  Sachs'  interpretation  as  already  stated  is  as  follows : 
The  bovine  sensitizer  unites  readily  with  the  corpuscles  as  soon  as 
its  affinity  for  alexin  (horse)  is  satisfied,  in  other  words,  when  its 
complementophilic  group  is  saturated.  This  is  the  reason  that 
hemolysis  takes  place  in  a  mixture  of  the  two  sera.  But  the  sen- 
sitizer shows  little  or  no  affinity  for  the  corpuscles  unless  previously 
combined  with  the  alexin.  This  explains  why  the  corpuscles  remain 
intact  when  treated  successively  with  ox  serum  and  then  with 
horse  serum.  If  these  interpretations  are  correct,  as  Ehrlich  and 
Sachs  affirm,  we  are  forced  to  admit  that  a  saturation  of  the  com- 
plementophilic group  by  means  of  alexin  increases  the  chemical 
affinity  of  the  cytophilic  group,  in  other  words,  the  affinity  for  the 
corpuscles. 

Theoretically,  it  is  rather  difficult  to  conceive  of  such  a  repercus- 
sion as  this,  since,  according  to  Ehrlich's  theory,  the  two  atom 
groups  are  distinct  and  independent.  It  is  better  at  all  events  to 
remain  within  the  bounds  of  experimentation.  The  experiments 
which  we  have  just  mentioned  are  the  only  ones  that  Ehrlich  and 
Sachs  have  referred  to.  As  we  shall  see,  it  would  have  been  prefer- 
able for  them  to  have  investigated  somewhat  further  and  to  have 
adduced  other  experiments  before  offering  their  interpretation. 

There  is  one  fact  in  particular  which,  although  remarkable  and 
certainly  of  significance  in  any  correct  interpretation,  seems  to  have 


370  STUDIES   IN   IMMUNITY. 

escaped  the  attention  of  these  writers.  Heated  bovine  serum  hus 
only  a  slight  agglutinating  property  for  guinea-pig  corpuscles. 
Fresh  horse  serum  does  agglutinate  them,  but  only  slowly  and  when 
present  in  relatively  large  amounts,  several  hours  may  be  required 
to  clump  the  corpuscles  in  any  considerable  masses.  A  mixture  of 
the  two  sera,  however,  agglutinates  in  a  very  few  minutes,  and  the 
corpuscles  soon  form  veritable  chunks  that  stick  to  the  glass.  We 
may  mention  in  this  connection  an  experiment  with  fresh  horse 
serum  (alexin),  bovine  serum  heated  to  56  degrees,  and  a  5  per 
cent  suspension  of  washed  guinea-pig  corpuscles;  mixtures  are 
prepared  as  follows: 

1.  Corpuscle  suspension,  1  c.c.;  bovine  serum,  56  degrees,  0.5 
of  a  cubic  centimeter. 

2.  Corpuscles,  1  c.c.;  horse  serum,  0.5  of  a  cubic  centimeter. 

3.  Corpuscles,  1  c.c.;  0.5  of  a  cubic  centimeter  of  a  mixture  in 
equal  parts  of  the  two  sera. 

There  is  intense  agglutination  in  3  in  a  few  minutes;  hemolysis 
begins  shortly  after  and  later  becomes  complete;  mixtures  1  and  2 
not  only  are  not  hemolyzed,  but  show  only  a  very  delayed  agglu- 
tination which  is  never  comparable  with  the  one  in  3. 

A  slight  variation  in  the  experiment  may  be  made  by  nrnking 
mixtures  1  and  2  and  then  mixing  them.  The  corpuscles  rapidly 
agglutinate  in  a  mixture  of  the  two  sera  and  are  soon  laked.*  It 
should  be  noted  at  once  that  if  horse  serum  heated  to  56  degrees 
is  added  in  place  of  fresh  horse  serum  to  the  heated  bovine  serum 
there  is  not  only  no  hemolysis,  but  also  none  of  this  intense  agglu- 
tination. The  agglutination,  then,  would  seem  in  some  way  depend- 
ent on  the  presence  of  active  alexin. 

This  is  a  rather  curious  condition.  If  an  explanation  were  sought 
according  to  Ehrlich  and  Sachs'  explanation,  we  should  have  to 
conclude  that  the  agglutinin  as  well  as  the  sensitizer  must  combine 
with  the  alexin  before  uniting  with  the  corpuscle.  Such  a  con- 
clusion is  unusual,  for  there  are  no  facts  that  would  lead  us  to  assume 
that  the  agglutinins  need  alexins  to  be  efficient. 

Ehrlich  and  Sachs'  theory  of  the  mode  of  action  of  these  two  sera 
seems  already  open  to  criticism.  We  must  therefore  consider  in 

*  This  agglutination  takes  place  at  room  temperature,  but  is  more  rapid  at 
37  degrees. 


RELATIONS  OF  SENSITIZERS  TO  ALEXIN.       .          371 

detail  the  logic  they  have  adopted  in  arriving  at  their  conclusion. 
Let  us  follow,  then,  their  argument  step  by  step :  First :  In  order 
to  prove  that  the  sensitizer  of  heated  bovine  serum  does  not  com- 
bine with  guinea-pig  corpuscles  when  horse  serum  is  absent,  these 
authors  rely  on  the  fact  that  corpuscles  treated  with  bovine  serum 
remain  intact  when  subjected  to  horse  alexin.  Such  an  argument 
is  valid  only  on  the  supposition  that  horse  alexin  is  capable  of  hemo- 
lyzing  sensitized  corpuscles.  Such,  however,  is  not  the  case.  Horse 
alexin,  indeed,  differs  from  the  alexins  of  most  sera  in  this  very 
respect.  Ehrlich  and  Morgenroth  have  themselves  noted*  that 
bovine  corpuscles,  sensitized  by  an  inactivated  hemolytic  serum 
from  the  rabbit,  are  not  hemolyzed  by  horse  alexin,  although  very 
small  doses  of  rabbit  or  guinea-pig  alexin  suffice  to  destroy  them. 
They  have  explained  the  fact  by  saying  that  the  horse  alexin  does 
not  "fit"  the  rabbit  sensitizer,  that  is  to  say,  fails  to  combine  with 
the  complementophilic  group  of  this  sensitizer;  in  other  words,  the 
alexin  is  not  absorbed  by  the  sensitized  cells.  It  may  be  noted, 
however,  that  this  latter  fact  is  not  true.,  for,  as  we  shall  later  see, 
the  alexin  does  become  fixed  by  such  corpuscles,  although  it  fails 
to  destroy  them.f 

There  is  no  proof,  then,  that  because  guinea-pig  corpuscles  treated 
by  bovine  serum  remain  intact  in  horse  alexin,  it  is  due  to  their 
not  being  sensitized,  since,  when  they  are  undoubtedly  sensitized, 
they  give  no  hemolysis  with  this  alexin.  If,  indeed,  we  take  guinea- 
pig  corpuscles,  sensitized  by  a  specific  inactivated  serum  from  the 
rabbit,  we  find  that,  although  they  are  hemolyzed  by  traces  of 
fresh  rabbit  serum  or  even  by  guinea-pig  serum,  they  remain  intact 
in  moderate  doses  of  horse  alexin  and  are  destroyed  by  large  doses 
only  after  a  long  period. 

It  is,  moreover,  easy  to  prove  that  there  is  a  moderately  powerful 
sensitizer  in  bovine  serum  acting  on  guinea-pig  corpuscles  in  the 
ordinary  way,  that  is,  by  uniting  with  them  when  no  alexin  is  present. 
A  few  tenths  of  a  cubic  centimeter  of  bovine  serum  (56  degrees) 
will  hemolyze  1  c.c.  of  a  5  per  cent  suspension  of  guinea-pig  cor- 

*  Ehrlich  and  Morgenroth,  Studies  on  Immunity,  Ehrlich-Bolduan,  John 
Wiley  &  Sons,  p.  88. 

t  This  fact  might  be  stated  in  the  Ehrlich  parlance  by  saying  that  horse  alexin 
has  no  toxophore  group  or  that  this  group  is  too  weak. 


372  STUDIES  IN  IMMUNITY. 

puscles  on  the  addition  of  guinea-pig  alexin  (0.3  of  a  cubic  centi- 
meter). No  hemolysis,  however,  occurs  if,  instead  of  ordinary 
heated  bovine  serum,  the  same  serum  previously  treated  with  guinea- 
pig  corpuscles  and  separated  from  them  by  centrifugalization  is 
used.  Such  corpuscles  have  absorbed  the  sensitizer. 

Second:  Ehrlich  and  Sachs  think  that  the  bovine  serum  (56 
degrees)  is  the  only  sensitizing  serum  in  their  experiment.  No 
proof  of  this  is  given,  and,  as  a  matter  of  fact,  the  horse  serum 
also  contains  a  sensitizer  for  guinea-pig  corpuscles  which  is  usually 
stronger  than  the  one  in  bovine  serum.  A  mixture  of  horse  serum 
(0.3  of  a  cubic  centimeter)  and  guinea-pig  corpuscles  (1  c.c.  of  a 
5  per  cent  suspension)  gives  hemolysis  on  the  addition  of  guinea- 
pig  alexin  (0.3  of  a  cubic  centimeter).  It  is  not  surprising  that 
fresh  horse  serum  alone  fails  to  hemolyze  the  blood,  although  it  con- 
tains both  alexin  and  sensitizer,  when  we  take  into  consideration, 
as  just  shown,  that  the  horse  alexin  fails  to  hemolyze  these  cor- 
puscles even  when  they  are  well  sensitized.  It  is  to  be  noted  in 
passing  that  the  horse  sensitizer  is  very  thermolabile,  being  almost 
entirely  deprived  of  its  power  by  heating  to  56  degrees. 

Third :  Ehrlich  and  Sachs  think  that  ox  serum  acts  only  as  a 
sensitizer  in  their  experiment. 

Is  it  true  that  the  bovine  serum  owes  its  entire  or  even  the 
greater  part  of  its  efficacy  to  its  sensitizing  property?  May  there 
not  also  be,  in  addition  to  the  sensitizer,  some  particular  substance 
in  bovine  serum  that  has  not  as  yet  been  described  in  this  or  in 
other  sera?  No  such  possibility  has  occurred  to  Ehrlich  and  Sachs, 
who  regard  the  bovine  serum  simply  as  containing  a  sensitizer.  It 
is  evident  that  to  decide  such  a  question  the  sensitizing  property  of 
the  serum  must  first  be  removed.  In  other  words,  we  must  have 
conditions  in  which  the  sensitizer  not  only  need  not  but  actually 
does  not  enter  into  consideration.  If,  under  such  conditions, 
heated  bovine  serum,  although  without  a  sensitizer,  hemolyzes  cor- 
puscles with  horse  alexin,  we  must  conclude  that  the  serum  con- 
tains some  other  as  yet  unsuspected  substance  that  is  of  capital 
importance  in  the  experiment. 

These  conditions  are  easy  to  fulfill  by  adding  well-sensitized 
bovine  blood  corpuscles*  instead  of  guinea-pig  corpuscles  to  a 

*  Previously  treated  with  immune  serum  from  the  rabbit  and  then  washed. 


RELATIONS  OF  SENSITIZERS  TO  ALEXIN.          ,         373 

mixture  of  heated  bovine  serum  and  horse  alexin.  It  is  evident  in 
such  an  experiment  that  not  only  is  a  sensitizer  in  the  bovine  serum 
unnecessary,  but  that  no  such  an  iso-  or  auto-sensitizer  can  exist.* 

We  take,  then,  bovine  corpuscles  that  have  been  well  sensitized 
by  immune  serum  from  the  rabbit.  On  the  addition  of  alexic 
horse  serum  neither  hemolysis  nor  agglutination  occurs,  f  But 
the  addition  of  the  horse  serum  plus  heated  bovine  serum  produces 
an  extreme  agglutination  followed  by  slow  but  distinct  hemolysis. 

The  details  of  this  experiment  are  as  follows: 

Well-washed  bovine  corpuscles  are  treated  with  three  volumes 
of  rabbit  antibovine  serum.  Two  or  three  hours  later  the  excess 
of  serum  is  removed  by  washing  in  salt  solution  and  the  superna- 
tant fluid  rejected.  Salt  solution  is  then  added  to  the  corpuscle 
sediment  so  as  to  make  a  20  per  cent  suspension.  Three  tubes  are 
made  as  follows: 

Tube  1.     Fresh  horse  serum,  0.3  c.c. 

Tube  2.    Bovine  serum  (56  degrees),  0.3  c.c. 

Tube  3.  Fresh  horse  serum,  0.3  c.c.  +  bovine  serum,  56  degrees, 
0.3  c.c. 

To  each  tube  is  then  added  0.3  of  a  cubic  centimeter  of  sensi- 
tized bovine  corpuscles.  As  a  result,  no  agglutination  or  hemoly- 
sis in  tubes  1  and  2;  rapid  agglutination  followed  by  hemolysis  in 
tube  3. 

Such  a  result  is  unexpected  and  paradoxical  in  view  of  current 
ideas  on  sera.  It  is  quite  conceivable  that  sensitized  ox  corpuscles 
should  remain  intact  in  horse  alexin,  for  we  know  that  the  alexins 
of  certain  species  are  too  weak,  or  are  unsuitable  for  hemolysis. 
But  the  fact  that  the  addition  of  the  very  serum  that  should  be  most 
inactive,  namely,  the  proper  serum  of  the  corpuscles  employed, 
should  destroy  the  corpuscles  is  at  least  peculiar.  This  serum, 
moreover,  has  lost  its  alexin  through  heating.  It  is  also  a  surprising 
fact  that  this  form  of  agglutination  requires  the  cooperation  of  two 
sera,  neither  of  which  alone  affects  the  corpuscles  in  question. 

The  analogy  between  this  experiment  and  the  one  described  by 

*  In  most  of  these  experiments  bovine  serum  and  corpuscles  from  the  same 
individual  were  employed. 

t  It  is  also  to  be  noted  that  the  rabbit  antibovine  serum,  although  highly 
sensitizing,  has  little  or  no  agglutinating  property. 


374  STUDIES   IN   IMMUNITY. 

Ehrlich  and  Sachs  is  evident.  In  their  experiments  guinea-pig  cor- 
puscles which  remain  intact,  either  in  heated  bovine  serum  or  in 
fresh  horse  serum,  are  destroyed  by  a  mixture  of  the  two.  In  our 
experiment  sensitized  ox  corpuscles  act  in  the  same  manner.  As 
far  as  agglutination  is  concerned  the  analogy  also  is  perfect.  It  is 
probable,  then,  that  both  experiments  are  subject  to  a  common 
explanation.  And,  what  is  more,  the  explanation  of  Ehrlich  and 
Sachs  would  already  seem  untenable. 

It  is  not  to  be  supposed  that  bovine  serum  contains  an  amboceptor 
that  unites  with  its  own  corpuscle  and,  on  the  other  hand,  with  the 
alexin  of  horse  serum,  or  that  this  combination  with  the  alexin  is 
necessary  to  produce  a  hypothetical  union  with  the  corpuscles.  A 
careful  study  of  the  facts,  moreover,  renders  such  a  supposition 
quite  impossible.  We  may  consider,  then,  in  detail  the  data  offered 
by  the  experiment  that  we  have  reported. 

a.  Is  it  necessary,  in  order  to  produce  agglutination  and  hemoly- 
sis  of  bovine  corpuscles  by  means  of  horse  serum  and  heated  bovine 
serum,  that  these  corpuscles  should  be  sensitized?    We  add  to  0.5 
of  a  cubic  centimeter  of  a  20  per  cent  emulsion  of  unsensitized 
corpuscles  0.3  of  a  cubic  centimeter  of  each  serum.    Nothing  occurs 
and  the  corpuscles  remain  intact.    Experiment  shows,  then,  that  the 
corpuscles  must  be  sensitized  in  order  to  be  agglutinated  and  de- 
stroyed, and  that  the  necessary  amboceptor  acting  upon  ox  corpuscles 
is  not  present  in  ox  serum.     Experiment  further  shows  that  horse 
serum  has  no  distinct  sensitizing  effect  for  bovine  blood  corpuscles. 

b.  The  presence  of  alexin  in  the  mixture  is  obviously  necessary 
for  hemolysis;  but  is  it  equally  indispensable  for  agglutination? 
We  add  0.3  of  a  cubic  centimeter  of  heated  bovine  serum  and  0.3  of 
a  cubic  centimeter  of  horse  serum  heated  to  56  degrees  to  0.5  of  a 
cubic  centimeter  of  a  suspension  of  sensitized  bovine  corpuscles. 
There  is  no  hemolysis  or  agglutination. 

c.  Does  the  horse  serum  simply  furnish  the  alexin  or  does  it 
also  furnish  the  principle  that  causes  agglutination?    If  it  only 
furnishes  the  alexin  it  may  evidently  be  replaced  by  any  other 
fresh  serum,  as,  for  example,  fresh  rabbit  serum.    This  proves  to 
be  the  case,  as  the  following  experiment  shows :  We  add  to  each  of 
two  tubes  0.5  of  a  cubic  centimeter  of  a  suspension  of  sensitized 
bovine  corpuscles  and  to  the  first  tube  add  0.2  of  a  cubic  centimeter 


RELATIONS  OF  SENSITIZERS  TO  ALEXIN.  375 

of  rabbit  alexin;  to  the  second  0.2  of  a  cubic  centimeter  of  rabbit 
alexin  plus  0.3  of  a  cubic  centimeter  of  heated  bovine  serum.  The 
corpuscles  in  both  tubes  hemolyze,  as  the  rabbit  alexin  is  strongly 
hemolytic  for  the  sensitized  corpuscles.  But  the  hemolysis  is 
preceded  in  the  second  tube  by  an  extraordinary  agglutination 
which  does  not  occur  in  the  first.  A  control  tube  shows  that  neither 
agglutination  nor  hemolysis  would  have  taken  place  if  an  emulsion 
of  normal  bovine  corpuscles,  instead  of  sensitized  bovine  corpuscles, 
had  been  employed.  It  is,  then,  the  bovine  serum  and  not  the  horse 
serum  that  furnishes  the  agglutinating  principle,  and,  as  we  have 
already  determined,  agglutination  takes  place  only  when  the  cor- 
puscles are  treated  both  with  a  sensitizer  and  with  an  alexin.  The 
origin  of  this  alexin,  however,  is  indifferent  and  may  be  either  from 
horse  serum,  rabbit  serum  or  even,  as  we  shall  see,  from  bovine 
serum  itself. 

We  add  to  each  of  two  tubes  0.2  of  a  cubic  centimeter  of  fresh 
non-heated  bovine  serum.  To  the  first  tube  we  then  add  0.5  of  a 
cubic  centimeter  of  normal  bovine  corpuscles  and  to  the  second  tube 
an  equal  amount  of  bovine  corpuscles  that  have  already  been  treated 
with  rabbit  antibovine  serum.  There  is  a  rapid  agglutination  of 
the  corpuscles  in  the  second  tube,  but  none  in  the  first. 

To  sum  up,  we  find  that,  in  presence  of  any  alexin,  bovine  serum 
will  agglutinate  corpuscles  of  the  same  species  (and  even  of  the 
same  animal)  provided  they  be  sensitized.  When  the  alexin  is 
only  weakly  hemolytic,  as  is  the  case  with  horse  alexin,  its  task  is 
greatly  facilitated  by  the  addition  of  bovine  serum.  In  other  words, 
this  serum  not  only  agglutinates  sensitized  corpuscles  that  have 
been  subjected  to  alexin,  but  changes  these  corpuscles  in  such  a 
way  that  alexin  that  might  otherwise  be  impotent  becomes  strong 
enough  to  produce  hemolysis.  This  explains  why  sensitized  bovine 
corpuscles  which  show  no  effect  when  subjected  to  horse  alexin  are 
agglutinated  and  hemolyzed  when  heated  bovine  serum  is  added.* 

Bovine  serum  causes  bovine  corpuscles  to  be  more  easily  destroyed 

*  It  is  to  be  noted  that  we  do  not  say  that  it  is  on  account  of  its  agglutination 
that  ox  serum  renders  corpuscles  more  accessible  to  the  action  of  a  weak  alexin. 
What  we  do  say  is,  that  bovine  serum  renders  corpuscles  more  accessible  to  alexic 
activity  and  in  addition  produces  a  very  marked  agglutination.  It  is  not  owing  to 
the  fact  that  the  corpuscles  are  clumped  that  they  become  more  susceptible  to  the 
alexin,  as  we  shall  later  see. 


376  STUDIES  IN   IMMUNITY. 

by  horse  alexin;  one  might  almost  say  that  it  increases  their  sen- 
sitivity. We  purposely  avoid  using  this  expression,  since  it  might 
lead  to  a  supposition  that  bovine  serum  contains  a  real  sensitizer 
for  its  own  corpuscles.  If  the  term  sensitization  were  loosely  used, 
it  might,  perhaps,  be  employed  in  this  case,  that  is  to  say,  if  it  were 
used  in  the  sense  of  rendering  the  corpuscles  more  susceptible  to 
destruction,  as  is  the  case  when  the  amount  of  sodium  chloride 
present  is  diminished;  the  word  sensitization,  however,  has  been 
used  in  a  more  exact  sense  by  one  of  us  for  some  time,  which  suffices 
to  prohibit  its  use  in  the  present  instance  as  applied  to  bovine 
serum.  We  know,  indeed,  that  the  true  sensitizers  do  not  affect 
their  proper  corpuscles  and,  on  the  other  hand,  that  corpuscles  that 
have  once  been  properly  sensitized  do  not  need  the  aid  of  another 
sensitizer  in  order  to  be  hemolyzed  by  alexin.  In  a  similar  way  it 
is  evident  that  this  substance  in  bovine  serum  should  not  be  con- 
fused with  the  ordinary  agglutinins,  although  it  does  lead  to  an 
agglutination. 

We  find,  in  fact,  only  one  logical  explanation  for  this  peculiar 
activity  of  bovine  serum;  and  in  order  to  elucidate  the  following 
discussion  we  may  announce  this  explanation  at  once,  although  it 
is  experimentally  proved  only  in  subsequent  pages.  We  believe 
that  there  exists  in  bovine  serum  a  peculiar  substance  that  resists 
heating  to  56  degrees  (and,  it  may  be  added,  is  retained  in  heated 
serum  for  months),  of  an  apparently  albuminous  and  colloidal 
nature,  which  shows  no  effect  on  red  blood  cells  so  long  as  they  are 
under  normal  conditions,  but  which  unites  with  them  as  soon  as 
they  are  laden  with  sensitizer  and  alexin.  We  have  to  deal,  we 
believe,  with  a  pure  phenomenon  of  molecular  adhesion.  From  the 
point  of  view  of  properties  of  molecular  adhesion  it  is  evident  that 
normal  corpuscles  differ  from  the  complex  that  results  in  a  mixture 
of  corpuscles,  sensitizer  and  alexin,  inasmuch  as  this  complex  has 
the  property  of  attracting  and  binding  to  it  this  substance  in  ox 
serum  that  the  normal  corpuscle  does  not  possess.  The  adhesion 
between  this  substance  and  sensitized  and  alexinized  corpuscles 
produces  their  agglutination  in  large  clumps  and  also  leads  to  a 
modification  in  them  which  renders  them  more  easily  hemolyzed 
by  alexins  of  moderate  potency. 

We  shall  refer  briefly  from  this  time  on  to  this  substance  in  bovine 


RELATIONS   OF  SENSITIZERS  TO  ALEXIN  377 

serum  that  attaches  itself  to  sensitized  and  alexinized  corpuscles 
as  " bovine  colloid." 

Before  we  endeavor  to  explain  the  accuracy  of  the  interpretation 
that  we  have  just  offered,  we  may  apply  it  to  the  hemolysis  of  guinea- 
pig  corpuscles,  since  these  are  the  corpuscles  that  are  used  in 
Ehrlich  and  Sachs'  experiment.  To  render  the  transition  between 
the  experiments  we  have  just  recounted  and  the  experiment  of 
Ehrlich  and  Sachs  more  simple,  we  may  deal  first  with  guinea-pig 
corpuscles  that  have  been  sensitized  in  the  same  manner  as  were 
our  bovine  corpuscles,  that  is  to  say,  with  a  specific  antiguinea- 
pig  serum  (a  serum,  heated  to  56  degrees,  from  a  rabbit  that  had 
been  immunized  against  guinea-pig  blood). 

These  sensitized  guinea-pig  corpuscles  are  hemolyzed  by  fresh 
guinea-pig  serum,  although  the  hemolysis  is  rather  slow,  particularly 
when  the  amount  of  alexin  employed  is  small,  owing  to  the  fact 
that  the  alexin  comes  from  the  same  animal  species.  Under  such 
conditions,  by  analogy  with  the  experiments  already  considered,  the 
addition  of  heated  bovine  serum  should  have  a  distinct  accelerating 
.action  on  the  hemolysis.  The  addition  of  this  serum,  moreover,  to 
these  sensitized  corpuscles  that  have  been  mixed  with  alexin  should 
bring  about  very  marked  agglutination. 

These  expectations  are  experimentally  confirmable.  Sensitized 
guinea-pig  corpuscles  are,  to  be  sure,  already  distinctly  agglutinated 
by  rabbit  antiguinea-pig  serum,  but  as  soon  as  the  alexin  and  heated 
bovine  serum  are  added  the  agglutination  becomes  much  more 
marked.  The  corpuscles  are  immediately  collected  into  large 
glistening  clumps  which  are  soon  hemolyzed.  The  bovine  serum 
produces  no  such  agglutination  with  the  sensitized  corpuscles  when 
no  alexin  is  present;  the  bovine  colloid,  we  repeat,  affects  corpus- 
cles only  when  they  have  been  both  sensitized  and  alexinized. 
The  analogy,  then,  between  this  experiment  and  the  one  with 
sensitized  ox  corpuscles  is  complete.  The  details  of  the  experi- 
ment follow:* 

*  It  is  to  be  noted  that  the  particular  rabbit  antiguinea-pig  serum  that  we 
have  used  for  this  experiment  was  obtained  by  injecting  rabbits  with  carefully 
washed  guinea-pig  red  blood  cells.  This  antiguinea-pig  serum  was  neither 
precipitating  nor  anti-alexic  for  guinea-pig  serum;  if  it  had  been,  the  experiment 
might  have  been  vitiated  by  a  neutralization  of  the  guinea-pig  alexin  in  the  tubes 
3  and  4  following. 


378  STUDIES  IN   IMMUNITY. 

The  following  mixtures  are  made: 

Tube  1.     Guinea-pig  alexin,  0.1  c.c.;  bovine  serum  (56  degrees),  0.3  c.c. 

Tube  2.  Rabbit  antiguinea-pig  serum  (56  degrees),  0.15  c.c.;  bovine  serum 
(56  degrees),  0.3  c.c. 

Tube  3.  Guinea-pig  alexin,  0.1  c.c.;  rabbit  antiguinea-pig  serum  (56  degrees), 
0.15  c.c.;  bovine  serum  (56  degrees),  0.3  c.c. 

Tube  4.  Guinea-pig  alexin,  0.1  c.c.;  rabbit  antiguinea-pig  serum  (56  degrees), 
0.15  c.c. 

Tube  5.     Rabbit  antiguinea-pig  serum,  0.15  c.c. 

To  each  tube  is  then  added  0.5  of  a  cubic  centimeter  of  a  5  per 
cent  suspension  of  washed  guinea-pig  blood.  At  room  temperature 
the  following  results  occur: 

In  tube  3,  in  which  the  bovine  serum  affects  sensitized  and  alex- 
inized  corpuscles,  a  very  powerful  agglutination  occurs  in  a  few 
moments,  and  hemolysis  is  complete  in  10  minutes.  In  mixture  4, 
which  is  identical  with  3  except  in  not  containing  bovine  serum, 
there  is  only  slight  agglutination,  and  hemolysis  is  incomplete  in 
half  an  hour.  Tubes  2  and  5  that  contain  no  alexin  give  a  slight 
agglutination,  but  no  hemolysis.  In  tube  1  that  contains  alexin, 
but  no  sensitizer,  the  corpuscles  are  not  agglutinated  and  show  only, 
a  very  slow  partial  hemolysis  which  is  incomplete  on  the  following 
day.  This  hemolysis  is  due,  as  we  have  already  seen,  to  the  fact 
that  heated  bovine  serum  contains  a  weak  sensitizer  for  guinea-pig 
corpuscles.* 

Now  that  we  have  determined  that  the  heated  bovine  serum 
acts  by  adhesion  of  the  colloidal  substance  on  sensitized  and  alex- 
inized  corpuscles  both  of  the  ox  and  of  the  guinea-pig,  Ehrlich  and 
Sachs'  experiment  is  readily  understood.  It  may  be  explained 
just  as  the  preceding  experiments,  namely,  by  supposing  that  in 
the  mixture  of  fresh  horse  serum  and  heated  bovine  serum  the 

*  This  mixture  containing  alexin  and  corpuscles  sensitized  simply  with  heated 
bovine  serum  (and  not  with  the  specific  antiserum)  is  not  sufficiently  sensitized 
or  fixed  with  alexin  to  bring  about  an  energetic  adhesion  of  the  colloid;  this  latter 
substance,  as  we  shall  later  see,  under  these  conditions  is  somewhat  absorbed,  so 
that  hemolysis  although  slight  is  increased. 

It  is  to  be  noted  that  the  weakness  of  the  bovine  sensitizer  is  due  perhaps  to 
heat.  This  substance  indeed  appears  to  be  more  powerful  in  unheated  bovine 
serum.  We  find  that  guinea-pig  corpuscles  are  energetically  clumped  by  fresh 
serum,  which  shows  that  the  colloid  has  adhered;  this  means,  of  course,  that  they 
have  been  well  sensitized  and  also  alexinized  by  the  fresh  bovine  serum;  hemolysis 
subsequently  occurs. 


RELATIONS  OF  SENSITIZERS  TO  ALEXIN.  379 

guinea-pig  corpuscles  become  laden  with  sensitizer  and  alexin  and 
are  therefore  susceptible  to  absorb  the  colloidal  substance  which 
brings  about  agglutination  and  facilitates  hemolysis. 

Which  of  the  two  sera  is  it  that  produces  the  sensitization?  We 
have  already  seen  that  heated  bovine  serum  contains  a  sensitizer 
inasmuch  as  its  addition  to  a  mixture  of  corpuscles  and  guinea-pig 
alexin  produces  a  distinct  hemolysis.  This  sensitizer  may  produce 
some  effect  in  the  experiment,  but  it  is  by  no  means  necessary, 
because  we  have  already  found  that  heated  bovine  serum  treated 
with  guinea-pig  corpuscles  and  thereby  deprived  of  its  sensitizer 
for  these  corpuscles  retains  intact  its  property  of  endowing  fresh 
horse  serum  with  a  hemolytic  property.  It  is,  moreover,  easy  to 
understand  why  the  bovine  sensitizer  is  not  necessary  inasmuch 
as  another  sensitizer  of  superior  potency  is  present  in  Ehrlich  and 
Sachs'  experiment.  This  is  the  sensitizer  contained  in  fresh  horse 
serum  in  addition  to  the  alexin.* 

Our  interpretation  of  Ehrlich  and  Sachs'  experiment  is  there- 
fore the  following:  When  guinea-pig  corpuscles  are  added  to  a 
mixture  of  the  two  sera  they  are  affected  by  the  sensitizer  of  the 
horse  serum  and  to  a  certain  extent  by  the  sensitizer  in  heated 
bovine  serum.  This  second  sensitizer  is,  however,  superfluous. 
Its  presence  is  by  no  means  necessary  for  the  experiment.  When 
this  sensitization  is  effected  the  corpuscles  are  then  in  a  condition 
to  fix  the  horse  alexin.  This  alexin,  however,  has  only  slight  hemo- 
lytic power.  But  once  the  corpuscles  have  become  sensitized  and 
laden  with  alexin,  they  are  modified  in  their  properties  of  molec- 
ular adhesion  to  such  an  extent  that  they  become  able  to  attract 
the  colloidal  substance  of  bovine  serum,  which  unites  with  them. 
The  adhesion  of  this  new  substance  produces  two  results :  It  causes 
the  blood  corpuscles  to  be  more  easily  destroyed  by  alexin  and 
also  agglutinates  them  energetically.  Consequently,  a  powerful 
clumping  followed  by  hemolysis  is  observed. 

This  interpretation  explains  all  the  facts  that  have  been  noted 
by  Ehrlich  and  Sachs.  It  is  evident,  for  example,  that:  (a)  bovine 
serum  (56  degrees)  that  has  been  treated  with  guinea-pig  corpuscles 
will  retain  its  property  of  forming  a  hemolytic  mixture  with  horse 

*  It  is  to  be  recalled  that  the  demonstration  of  this  sensitizer  is  easy.  Guinea- 
pig  corpuscles  are  hemolyzed  by  a  mixture  of  guinea-pig  alexin  and  horse  serum. 


380  STUDIES  IN   IMMUNITY. 

serum.  The  guinea-pig  corpuscles,  in  other  words,  may  remove 
all  the  sensitizer  from  the  bovine  serum,  since  this  substance  is  not 
necessary  in  the  hemolysis,  but  they  cannot  remove  the  colloidal 
substance.  This  colloidal  substance  is  absorbed  only  by  corpuscles 
that  have  been  treated  both  with  sensitizer  and  alexin. 

(6)  These  treated  guinea-pig  corpuscles  that  have  fixed  the 
sensitizer  but  not  the  colloidal  substance  of  bovine  serum  are  not 
hemolyzed  if  the  excess  of  serum  is  removed  and  horse  serum  added. 
Such  corpuscles,  although  they  are  sensitized  and  fix  the  alexin  in 
the  horse  serum,  are  not  subjected  to  the  effect  of  the  colloidal  sub- 
stance, which  has  been  removed  in  removing  the  excess  of  bovine 
serum. 

Inasmuch  as  the  preceding  experiments,  and  in  particular  those 
dealing  with  bovine  corpuscles,  have  shown  that  Ehrlich  and  Sachs' 
interpretation  formulated  on  purely  theoretical  and  preconceived 
grounds  is  inacceptable,  we  feel  scarcely  called  upon  to  consider  it 
further.  It  is  well,  however,  to  verify  the  accuracy  of  our  own 
explanation. 

First :  We  think  that  the  fixation  of  the  colloidal  substance  occurs 
only  when  the  corpuscles  have  been  treated  with  sensitizer  and 
alexin.  If  this  be  true  we  should  expect  that  corpuscles  so  sen- 
sitized and  alexinized  and  subsequently  carefully  washed  in  salt 
solution  would  absorb  the  colloidal  substance  of  heated  bovine 
serum  added  to  it.  We  should  not  expect  the  presence  of  free 
alexin  to  be  necessary  for  this  absorption. 

Inasmuch  as  clumping  is  a  visible  symptom  of  the  absorption 
of  the  colloid,  we  should  expect  sensitized,  alexinized  and  sub- 
sequently washed  corpuscles  to  show  this  phenomenon  on  the 
addition  of  heated  bovine  serum.  In  such  an  experiment -the  sen- 
sitized corpuscles  must  of  necessity  not  be  destroyed  by  the  alexin, 
and  for  this  purpose  horse  serum  which  has  only  slight  hemolytic 
properties  is  best. 

We  prepare,  then,  a  10  per  cent  suspension  of  ox  blood  that 
has  been  sensitized  with  a  specific  serum  from  the  rabbit  and 
subsequently  washed,  and  also  a  10  per  cent  suspension  of  non- 
sensitized  ox  blood.  The  following  mixtures  are  made  in  three 
large  tubes: 


RELATIONS   OF  SENSITIZERS  TO   ALEXIN.       .  381 

(a)  Suspension  of  sensitized  corpuscles,  1  c.c. ;  fresh  horse  serum, 
0.3  c.c. 

(b)  Sensitized  corpuscles,  1  c.c. ;  horse  serum,  56  degrees,  0.3  c.c. 

(c)  Non-sensitized  corpuscles,  1  c.c.;  fresh  horse  serum,  0.3  c.c. 
Half  an  hour  later  the  tubes  are  rilled  with  salt  solution,  cen- 

trifugalized  and  the  supernatant  fluids  decanted;  this  washing  is 
repeated.  After  the  second  decanting  1  c.c.  of  salt  solution  plus 
0.4  c.c.  of  bovine  serum  (56  degrees)  is  added  to  each  sediment  of 
corpuscles.  The  result  is,  no  agglutination  in  tubes  "6"  and  "c," 
but  a  strong  and  almost  instantaneous  agglutination  in  tube  "a." 

Second :  We  have  just  said  that  the  colloidal  substance  is  absorbed 
by  sensitized  and  alexinized  corpuscles.  If  this  is  true,  heated 
bovine  serum  that  has  been  treated  with  such  corpuscles  should 
not  be  able  to  agglutinate  fresh  corpuscles.  Experiment  verifies 
this  expectation.  Well-sensitized  bovine  corpuscles  are  washed, 
restored  to  primitive  volume  and  mixed  with  three  parts  of  fresh 
horse  serum.  Two  hours  later  the  corpuscles  are  washed  again, 
restored  to  primitive  volume  and  an  equal  amount  of  heated  bovine 
serum  is  added.  The  bovine  serum  is  found,  subsequent  to  this 
contact,  to  retain  little  if  any  of  its  colloidal  substance.* 

Third :  Bovine  serum  that  has  lost  the  greater  part  of  its  colloidal 
substance  in  this  manner  should  show  very  much  less  activity  than 
intact  serum  when  mixed  with  fresh  horse  serum  and  guinea-pig 
corpuscles.  We  find,  indeed,  that  agglutination  and  hemolysis 
of  these  corpuscles  are  very  much  less  in  the  presence  of  this  ex- 
hausted serum  than  with  a  corresponding  dose  of  untreated  bovine 
serum  or  with  bovine  serum  that  has  been  treated  simply  with 
guinea-pig  corpuscles  and  has  thereby  lost  its  sensitizer  only. 

Fourth:  The  colloidal  substance  is  absorbed  and  produces 
agglutination  only  when  the  corpuscles  are  alexinized  as  well  as 
sensitized.  In  the  Ehrlich  and  Sachs'  experiment  it  is  evidently 
horse  serum  that  furnishes  the  alexin.  We  should  expect,  then,  that 
if,  to  a  mixture  of  guinea-pig  corpuscles  and  heated  bovine  serum, 
horse  serum  that  has  been  treated  with  any  sensitized  corpuscles 

*  It  is  to  be  noted  that  very  small  amounts  of  colloidal  substance  suffice  to 
agglutinate  sensitized  and  alexinized  corpuscles.  A  complete  removal  of  the 
colloidal  substance  would  therefore  be  necessary  to  bring  about  an  entire  loss  of 
agglutinating  property. 


382  STUDIES  IN   IMMUNITY. 

is  added,  the  corpuscles  would  show  neither  hemolysis  nor  aggluti- 
nation. Experimentally  we  find  this  to  be  true.  Bovine  blood  is 
sensitized  with  three  volumes  of  serum  from  an  immunized  rabbit. 
The  corpuscles  are  then  washed,  the  original  volume  of  the  blood 
reestablished  and  an  equal  amount  of  fresh  horse  serum  added. 
After  a  time  this  mixture  is  centrifugalized  and  the  supernatant 
fluid  is  employed  as  horse  serum  deprived  of  alexin.  As  a  control 
we  use  horse  serum  that  has  been  treated  in  exactly  the  same  man- 
ner with  unsensitized  bovine  corpuscles.  The  following  mixtures 
are  then  prepared: 

(a)  5  per  cent  suspension  of  guinea-pig  corpuscles,  1  c.c. ;  bovine 
serum  (56  degrees),  0.3  c.c.;  treated  horse  serum,  0.6  c.c. 

(b)  Corpuscle  suspension,   1  c.c.;   bovine  serum  (56  degrees), 
0.3  c.c.;  control  horse  serum,  0.6  c.c. 

(c)  Corpuscle  suspension,   1  c.c.;   bovine  serum  (56  degrees), 
0.3  c.c.;  a  mixture  of  equal  parts  of  salt  solution  and  fresh  horse 
serum,  0.6  c.c. 

As  a  result, agglutination  appears  rapidly  in  "b" and  "c,"  followed 
by  hemolysis.  There  is  no  agglutination  or  hemolysis  in  "a." 

The  same  experiment  with  sensitized  ox  corpuscles  in  place  of 
guinea-pig  corpuscles  gives  a  similar  result. 

It  is  to  be  noted  in  passing  that  this  experiment  also  furnishes  a 
proof  of  the  functional  unity  of  the  alexin.  Horse  serum  that  has 
been  deprived  of  alexin  for  bovine  corpuscles  has  likewise  lost  its 
alexic  activity  for  guinea-pig  corpuscles.  Both  species  of  cor- 
puscles, therefore,  are  sensitive  to  the  same  alexin. 

Fifth :  According  to  our  idea,  in  Ehrlich  and  Sachs'  experiment  the 
fresh  horse  serum  not  only  furnishes  the  alexin  for  the  guinea-pig 
corpuscles,  but  is  also  of  prime  importance  in  sensitizing  them. 
Guinea-pig  corpuscles  that  have  been  placed  in  contact  with  fresh 
horse  serum  alone,  and  then  washed  and  so  deprived  of  the  excess 
of  serum,  should  be  both  sensitized  and  alexinized,  in  other  words, 
able  to  fix  the  colloidal  substance;  they  should  therefore  be  agglu- 
tinated on  the  addition  of  heated  bovine  serum,  whereas  normal 
guinea-pig  corpuscles  will  show  no  such  effect.  We  find  this,  experi- 
mentally, to  be  true.  As  a  control,  it  is  shown  that  corpuscles  treated 
in  the  reverse  manner,  that  is  to  say,  by  heated  bovine  serum  first, 
washed,  and  then  horse  serum,  show  no  agglutination. 


RELATIONS  OF  SENSITIZERS  TO  ALEXIN.  383 

Tube  a.  Ten  per  cent  suspension  of  guinea-pig  corpuscles, 
1  c.c. 

Tube  b.  Ten  per  cent  suspension  of  corpuscles,  1  c.c.;  fresh 
horse  serum,  0.3  c.c. 

Tube c.  Suspension  of  corpuscles,  1  c.c.;  bovine  serum  (56  de- 
grees), 0.3  c.c. 

An  hour  later  the  tubes  are  filled  with  salt  solution,  the  mixtures 
well  shaken  and  centrifugalized.  The  supernatant  fluids  are  then 
decanted  and  1  c.c.  of  salt  solution  is  added  to  each  sediment.  To 
tubes  "a"  and  "b"  is  then  added  0.3  of  a  cubic  centimeter  of  bovine 
serum  (56  degrees),  and  to  tube  "c"  0.3  of  a  cubic  centimeter  of 
fresh  horse  serum.  As  a  result,  there  is  a  powerful  and  almost 
immediate  agglutination  in  "6,"  but  nothing  of  the  kind  in  "a" 
or  "c." 

Sixth:  According  to  the  preceding  results  we  should  expect  that 
horse  serum  that  has  been  treated  with  guinea-pig  corpuscles  would 
be  deprived  both  of  its  sensitizer  and  of  its  alexin  for  these  cells,  and 
that  consequently  it  would  have  no  power  to  form  an  agglutinating 
and  hemolytic  mixture  for  fresh  guinea-pig  corpuscles  with  bovine 
serum.  This  exhaustion  of  horse  serum  by  contact  with  guinea- 
pig  corpuscles  is  easily  verified  experimentally,  as  Klein*  has  already 
shown.  This  fact,  moreover,  is  quite  irreconcilable  with  Ehrlich  and 
Sachs'  explanation.  There  is  one  fact,  however,  that  must  be 
noted  in  performing  this  experiment.  The  absorption  of  the  active 
substances  from  horse  serum  takes  place  perfectly  if  the  serum  is 
mixed  with  a  suspension  of  corpuscles,  that  is  to  say,  corpuscles 
sufficiently  diluted  in  salt  solution.  But  if  the  salt  solution  is 
removed,  that  is,  if  a  sediment  of  corpuscles  is  employed,  the  absorp- 
tion is  incomplete  and  the  serum  retains  the  larger  part  of  its 
original  properties.  This  fact  has  also  been  noted  by  Klein.  This 
would  show  that  the  fixation  of  active  principles  in  a  serum  is  in- 
fluenced by  very  slight  variations,  the  importance  of  which  well 
may  be  overlooked,  and,  consequently,  the  usual  method  of  specific 
absorption  as  employed  by  Ehrlich  and  his  followers  in  the  study 
of  hemolysis  may  lead  to  erroneous  conclusions.  In  the  present 
example,  for  instance,  one  might  be  led  to  conclude  that  the  cor- 

*  Ueber  die  Beeinflussung  des  hamolytischen  Komplements  durch  Agglutina- 
tion und  Prazipitation.  Wiener  klinische  Wochenschrift,  1905,  No.  48. 


384  STUDIES  IN  IMMUNITY. 

puscles  did  or  did  not  possess  receptors  for  the  active  substances 
of  horse  serum  in  accordance  with  whether  salt  solution  was  or  was 
not  present.  One  of  us  and  also  Landsteiner  have  already  noted 
the  causes  of  error  in  experiments  in  specific  absorption  of  this 
nature.  The  following  experiment  will  illustrate  this  point: 

Tube  a.  Fresh  horse  serum,  0.4  c.c.;  washed  guinea-pig  blood, 
40  per  cent  suspension  in  salt  solution,  1  c.c. 

Tube  6.  Fresh  horse  serum,  0.4  c.c.;  the  sediment  of  corpuscles 
derived  from  1  c.c.  of  40  per  cent  suspension. 

The  tubes  are  centrifugalized  after  an  hour's  contact  and  the  su- 
pernatant fluids  "a"  and  "b"  are  poured  into  tubes  "aa,"  which 
contain  0.05  c.c.  of  pure  guinea-pig  blood,  and  "66,"  which  contain 
this  amount  of  blood  plus  1  c.c.  of  salt  solution.  To  each  tube 
"oa"  and  "66"  is  then  added  0.4  c.c.  of  bovine  serum  (56  degrees). 
As  a  result,  there  is  no  agglutination  or  hemolysis  in  tube  "oa," 
but  in  tube  "66,"  on  the  contrary,  agglutination  and  hemolysis  are 
almost  as  rapid  and  powerful  as  in  a  control  mixture  containing 
0.05  c.c.  of  blood,  1  c.c.  of  salt  solution,  0.04  c.c.  of  horse  serum  and 
0.4  c.c.  of  bovine  serum  (56  degrees). 

We  then  take  the  sediments  in  tubes  "a"  and  "6"  respectively, 
and  add  to  each  1  c.c.  of  salt  solution  plus  1  c.c.  of  heated  bovine 
serum.  The  corpuscles  in  "6"  show  only  slight  and  very  gradual 
agglutination;  the  corpuscles  in  "a"  are  much  more  rapidly  agglu- 
tinated, which  means  that  they  have  absorbed  the  colloidal  sub- 
stance better,  and  are  better  sensitized  and  alexinized. 

In  the  same  way,  as  we  find  that  the  active  substances  of  horse 
serum  are  more  easily  absorbed  in  the  presence  of  salt  solution, 
we  also  find  that  agglutination  and  hemolysis  of  guinea-pig  cor- 
puscles in  a  mixture  of  bovine  serum  plus  horse  serum  occur  better 
when  the  mixture  contains  a  certain  amount  of  physiological  solu- 
tion than  when  it  does  not. 

Seventh:  In  Ehrlich  and  Sachs'  experiment  the  sensitization  of 
the  corpuscles  is  essentially  brought  about,  as  we  have  already  seen, 
by  horse  serum.  This  serum  would  naturally  lose  its  alexin  when 
added  to  sensitized  bovine  corpuscles,  and  would  keep  its  sensitizer 
for  guinea-pig  blood. 

If  we  should  add,  then,  to  such  a  serum  alexin  in  the  form  of  fresh 
guinea-pig  serum,  it  should  recover  its  property  to  hemolyze  guinea- 


RELATIONS   OF  SENSITIZERS  TO  ALEXIN.  385 

pig  corpuscles,  and  the  property  would  become  more  energetic  if 
one  added  in  addition  heated  bovine  serum. 

To  make  the  experiment  more  demonstrative,  the  bovine  serum 
may  first  be  deprived  of  its  sensitizer  for  guinea-pig  corpuscles  by 
contact  with  them;  in  this  case  its  action  is  due  entirely  to  its  col- 
loidal substance,  the  sensitizer  being  furnished  by  the  horse  serum. 

Under  these  conditions  a  mixture  of  guinea-pig  corpuscles, 
bovine  serum  (56  degrees)  that  has  been  treated  with  an  equal 
volume  of  guinea-pig  corpuscles,  fresh  horse  serum  that  has  been 
treated  with  an  equal  volume  of  sensitized  bovine  corpuscles,  and 
guinea-pig  alexin,  gives  a  marked  hemolysis. 

Proper  controls  show  that,  in  this  experiment,  if  bovine  serum  is 
not  present  the  hemolysis  by  the  treated  horse  serum  plus  guinea- 
pig  alexin  is  much  slower,  and  also,  in  another  control,  that,  if  horse 
serum  is  not  present,  treated  bovine  serum  plus  alexin  from  the 
guinea-pig  produces  no  hemolysis. 

Eighth:  One  final  remark  on  the  hemolysis  of  guinea-pig  corpuscles 
by  heated  bovine  serum  plus  guinea-pig  alexin  occurs  to  us.  It 
is  evident  that  in  such  a  mixture  the  colloidal  substance  may  take 
a  certain  additional  part,  inasmuch  as  the  corpuscles  are  some- 
what sensitized  by  the  bovine  serum,  and  therefore  capable  of  fixing 
the  alexin  and  subsequently  of  absorbing  a  certain  amount  of  colloid. 
This  latter  substance,  as  we  know,  tends  to  increase  hemolysis. 

Under  these  conditions  the  corpuscles  should  be  more  actively 
destroyed  than  when  they  have  been  first  sensitized  by  addition  of 
bovine  serum  and  then  washed  and  guinea-pig  alexin  subsequently 
added.  If  we  proceed  in  this  manner  we  suppress  the  colloid 
absorption  by  sensitized  and  alexinized  corpuscles.  In  other  words, 
we  find  experimentally  that  hemolysis  is  better  when  the  bovine 
serum  is  left  with  the  corpuscles  and  the  alexin  than  when  added 
to  the  corpuscles,  and  the  excess  eliminated  before  the  fresh  guinea- 
pig  serum  is  added. 

Of  course  no  such  peculiarity  occurs  in  the  hemolysis  of  corpuscles 
in  presence  of  alexin  and  horse  serum.  This  latter  serum,  as  we 
know,  acts  only  as  a  sensitizer  and  shows  none  of  the  properties  of 
the  bovine  colloid. 

The  interpretation  of  Ehrlich  and  Sachs*  experiment  that  we 
have  offered  is  therefore  experimentally  confirmable.  There  is, 


386  STUDIES  IN  IMMUNITY. 

however,  one  fact  that  at  first  sight  might  seem  not  to  agree  with 
our  explanation. 

When  guinea-pig  corpuscles  are  treated  with  a  sufficient  amount 
of  fresh  horse  serum,  washed,  and  then  added  to  heated  bovine 
serum,  an  energetic  agglutination  occurs,  due  to  the  absorption  of 
the  colloidal  substance  by  sensitized  and  alexinized  corpuscles. 
It  would  also  seem  as  if  under  these  conditions  an  active  hemolysis 
should  take  place,  since  we  know  that  hemolysis  is  also  favored  by 
the  addition  of  colloid.  The  hemolysis  under  these  conditions, 
however,  is  very  slow  and  slight,  although  the  agglutination  is  rapid 
and  strong. 

It  is  to  be  noted  that  in  such  an  experiment  the  alexin  and  colloid 
are  fixed  successfully  and  at  relatively  long  intervals  apart.  It 
would  seem  as  if  the  fact  that  the  alexin  had  been  acting  for  a  long 
time  had  increased  the  properties  of  molecular  adhesion  on  the 
part  of  the  corpuscles  to  its  maximum,  and  as  if  the  exaggerated 
absorption  of  the  colloid  had  brought  about  an  unusually  intense 
agglutination.  In  other  words,  it  would  seem  as  if  there  were  some 
antagonism  between  hemolysis  and  agglutination.  It  would  seem 
as  if,  when  agglutination  is  too  powerful,  the  corpuscles  would,  so 
to  speak,  coagulate,  and  so  become  more  resistant  to  alexin.  The 
proof  of  this  supposition,  which  incidentally  brings  out  the  fact  that 
absence  of  hemolysis  in  the  case  in  point  in  no  way  disagrees  with 
our  interpretation,  may  be  experimentally  shown  in  the  following 
manner:  Corpuscles  that  have  been  subjected  to  horse  serum  and 
subsequently  to  a  strong  agglutination  by  heated  bovine  serum  will 
resist  hemolysis  on  the  subsequent  addition  of  both  horse  serum 
and  bovine  serum,  in  other  words,  when  subjected  to  a  mixture 
that  is  highly  hemolytic  for  ordinary  corpuscles.  The  agglutinated 
corpuscles,  then,  have  a  greater  resistance  than  the  normal  corpuscles. 

In  short,  the  hemolysis  due  to  these  three  substances  is  a  phe- 
nomenon dependent  on  the  manner  in  which  the  various  factors 
are  added,  and  its  mechanism  is  so  delicate  as  to  be  easily  destroyed 
by  varying  the  experimental  conditions. 

The  reverse  of  this  condition  may  be  produced  also,  that  is  to 
say,  an  active  hemolysis  accompanied  by  a  slight  agglutination.  To 
increase  this  hemolytic  tendency  a  small  amount  of  guinea-pig 
alexin  is  added  to  the  mixture  of  bovine  serum  plus  horse  serum. 


RELATIONS  OF  SENSITIZERS  TO  ALEXIN.  387 

For  example,  hemolysis  is  much  more  rapid  in  a  mixture  of  1  c.c. 
of  5  per  cent  guinea-pig  blood  plus  0.3  of  a  cubic  centimeter  of 
heated  bovine  serum  plus  0.3  of  a  cubic  centimeter  of  horse  serum 
plus  0.3  of  a  cubic  centimeter  of  fresh  guinea-pig  serum  than  when 
the  guinea-pig  alexin  is  omitted.  In  the  first  instance  the  agglu- 
tination is  little  or  none,  whereas  in  the  second  it  is  strong. 

Hemolysis  and  agglutination,  therefore,  are  not  inseparable  and 
interdependent,  but  are  two  distinct  results  of  a  single  phenomenon, 
namely,  the  absorption  of  the  colloid  by  sensitized  and  alexinized 
corpuscles. 

CONCLUSIONS. 

1.  As  we  know  from  Ehrlich  and  Sachs'  experiment,  guinea- 
pig  corpuscles  are  hemolyzed  by  a  mixture  of  fresh  horse  serum  and 
heated  bovine  serum  (56  degrees),  but  they  resist  hemolysis  when 
they  are  treated  first  with  bovine  serum,  and  the  horse  serum  is 
subsequently  added.     The  interpretation  of  Ehrlich  and  Sachs, 
which  supposes  that  the  sensitizer  in  bovine  serum  will  not  unite 
with  corpuscles  until  it  has  first  combined  with  the  alexin,  is  inex- 
act.    In  the  first  place  the  more  powerful  and  necessary  sensitizer  is 
not  in  the  bovine  serum,  but  in  the  horse  serum.    Both  these  sensi- 
tizers,  moreover,  act  as  do  other  sensitizers  in  that  they  do  not  need 
alexin  in  order  to  unite  with  corpuscles.    In  their  interpretation, 
moreover,  Ehrlich  and  Sachs  make  no  mention  of  an  essential  factor 
which  gives  rise  to  the  remarkable  and  peculiar  appearance  of  the 
hemolysis  in  question. 

2.  This  factor  is  a  substance  present  in  bovine  serum  that 
resists  heat  to  56  degrees  and  may  be  kept  for  some  time.    It  is 
colloidal  in  nature,  and  is  doubtless  some  albuminous  substance 
which  has  the  property  of  uniting  with  corpuscles  that  are  laden 
with  sensitizer  and  alexin  and  which  remains  free  in  the  presence  of 
normal  or  of  simply  sensitized  corpuscles.    This  explains  why  the 
guinea-pig  corpuscles  not  only  are  destroyed  by  a  mixture  of  the 
two  sera,  but  the  reason  of  their  agglutination  in  voluminous  masses. 

3.  The  absorption  of  this  colloid  by  sensitized  and  alexinized 
corpuscles  is  probably  due  to  molecular  adhesion  due  to  a  change 
in  the  corpuscles  brought  about  by  the  two  substances  with  which 
they  have  been  treated.     If  both  sensitizer  and  alexin  are  present, 


388  STUDIES  IN   IMMUNITY. 

corpuscles  of  any  kind,  and  even  from  the  same  individual  that 
furnishes  the  colloid,  give  the  phenomenon. 

4.  The  presence  of  this  colloid  explains  the  peculiarities  that 
have  been  noted  by  Ehrlich  and  Sachs.    It  is,  moreover,  most  evident 
in  the  experiments  which  we  have  detailed  for  the  purpose  of  out- 
lining the  mode  of  action  of  the  various  substances  that  intervene 
in  the  agglutination  and  hemolysis  under  consideration. 

5.  Inasmuch  as  Ehrlich  and  Sachs'  interpretation  is  false,  it  is 
evident  that  the  single  argument  that  would  seem  to  prove  Ehrlich 's 
thesis  of  a  separate  complementophilic  atom  complex  in  the  sen- 
si  tizer  must  be  rejected.    And, accordingly, the  terms  " amboceptor  " 
and  "  complement "  should  be  abandoned  as  erroneous. 


XX.    ON  THE  NATURE  OF  OPSONINS  * 

BY  J.   G.  SLEESWIJK   (Leyden). 

In  a  previous  article  I  have  reported  some  experiments  bear- 
ing on  the  function  of  the  so-called  opsonins  in  cellular  immunity.! 
I  demonstrated  a  thermolabile  substance  in  frog  serum  which 
appears  indispensable  for  phagocytosis  of  the  anthrax  bacillus. 

I  have  since  continued  my  studies  on  this  subject  for  the  purpose 
of  answering  the  familiar  question:  Are  the  opsonins  individual 
substances  sui  generus,  or  are  they  identical  with  known  immune 
bodies?     I  may  refer  to  a  few  of  the  many  publications  on  opsonins 
which  are  of  particular  importance  in  this  connection. 

It  may  be  stated  at  once  that  my  work  has  led  to  the  conclusion 
that  the  non-specificity  or,  better,  non-autonomy  of  the  opsonins 
seems  to  me  fully  established. 

The  subject,  indeed,  is  far  from  new.  Metchnikoff  and  his  pupil 
Bordet  in  his  studies  on  preventive  sera,J  and  later  Savtchenko  § 
with  hemolysis,  and  indeed  all  investigators  that  have  worked  with 
antisera,  have  noted  their  favoring  influence  on  phagocytosis. 
The  explanations  of  this  influence  have,  however,  varied:  some 
have  thought  that  the  bacteria  are  prepared  for  the  action  of 
an  intracellular  or  an  extracellular  alexin  by  specific  sensitizers, 
whereas  Metchnikoff  assumes  the  existence  of  stimulins  with  a 
direct  influence  on  the  leucocytes.  || 

For  a  long  time  attempts  have  been  made  to  find  specific  sub- 
stances which  act  as  intermediaries  between  bacteria  and  phagocytes, 
since  we  know  that  there  are  preventive  sera  which  have  not  the 

*  Ueber  den  Bau  der  Opsonine.     Cent,  f.,  Bakt.,  I.  Orig.,  XLVI,  1908,  513. 
t  Annales  de  1'Institut  Pasteur,  December,  1907. 
J  On  the  mode  of  action  of  preventive  sera,  p.  81. 
§  Savtchenko.     Annales  de  1'Institut  Pasteur,  1902. 

II  Contrary  to  the  opinion  of  certain  investigators  (Sauerbeck,  Leishman), 
Metchnikoff  still  supports  hie  stimulin  theory.    Compare  Lubarsch  and  Ostertags' 
Ergebnisse,  1907. 

389 


390  STUDIES   IN   IMMUNITY. 

slightest  bactericidal  action  in  vitro,  but  act  particularly  as  stimu- 
lants to  phagocytosis. 

Denys  and  Leclef  *  showed  that  this  was  the  case  with  anti- 
streptococcus  serum,  and  Neufeld  and  Rimpau  t  later  corroborated 
these  findings  for  the  streptococcus  and  extended  them  to  the 
pneumococcus.  They  called  these  substances,  which  they  supposed 
to  exist  in  the  immune  sera  and  which  they  believed  to  act  favorably 
on  phagocytosis,  bacteriotropins. 

But  even  before  that  time  Wright,  working  with  Douglas  and 
several  other  collaborators,  had  perfected  a  special  technic  by  which 
it  was  possible  to  study  the  relations  of  phagocytes  to  bacteria  during 
the  process  of  phagocytosis.  He  demonstrated  in  the  blood  and 
in  other  body  fluids  substances  which  he  called  opsonins.  These 
substances  prepare  the  bacteria  for  phagocytosis  by  becoming  fixed 
upon  them.  They  are,  in  general,  thermolabile,  greatly  increased 
in  the  immune  sera,  and  specific. 

These  opsonins,  which  are  considered  individual  substances  by 
some,  are  by  others  believed  to  be  identical  with  the  sensitizers 
or  with  the  alexin.  Recently  LevaditiJ  with  two  collaborators, 
Inmann  and  Koessler,  assumed  an  intermediate  position  in  that  he 
believes  the  opsonins  of  normal  serum  to  be  identical  with  alexin 
and  the  opsonins  of  specific  sera  to  be  identical  with  the  sensitizers. 
Neufeld  and  Hiine§  maintain,  on  the  other  hand,  a  distinctive  posi- 
tion for  their  bacteriotropins.  Muir  and  Martin,  ||  in  a  series  of 
experiments,  using  a  technic  which  partially  corresponds  to  that  of 
my  experiments  (fixation  of  complement),  have  shown  a  marked 
similarity  between  alexin  and  opsonin. 

Let  us  now  consider  what  our  own  investigations  have  taught 
us.  I  wish  first  to  report  on  the  character  of  the  opsonins  in  normal 
sera.  In  my  first  experiments  I  employed  frog  leucocytes,  since 
I  knew  that  after  three  washings  with  physiological  saline  solution 
they  lose  completely  their  power  of  ingesting  anthrax  bacilli,  and 
that  fresh  serum  will  reactivate  them  by  the  fixation  of  the  serum 
opsonin  upon  the  bacteria.  Furthermore,  I  had  previously  con- 

*  La  Cellule,  1895. 

t  Deutsche  med.  Woch.,  1904,  and  Zeitschr.  f.  Hyg.  u.  Infektionskrank.  1905. 

t  C.  R.  Soc.  de  Biologic,  April  and  May,  1907. 

§  Arb.  a.  d.  Kaiserl.     Gesundheitsamte,  1907. 

||  Brit.  Med.  Journal,  December,  1906. 


ON   THE  NATURE  OF  OPSONINS.  391 

vinced  myself  of  the  fact  that  fresh  frog  serum  produces  com- 
pletely hemolysis  of  bovine  blood  corpuscles  previously  treated 
with  an  inactivated  specific  serum  (rabbit  >  bovine).  If,  then,  the 
anthrax  opsonin  of  frog  serum  were  identical  with  the  alexin,  the 
latter,  after  having  been  fixed  on  bacteria,  should  no  longer  be 
capable  of  hemolyzing  sensitized  red  blood  corpuscles.  The  entire 
experiment  with  the  controls  was  arranged  as  follows: 

A.  Into  each  of  6  very  small  tubes  a  drop  of  thick  emulsion 
of  anthrax  bacilli  (24-hour  agar  culture)  was  placed,  together  with 
ifo,  ?V,  *V,  T<J<T,  T??(T  and  ?$0  of  a  cubic  centimeter  of  fresh  frog 
serum.     These  were  properly  mixed,  left  for  one-half  hour  in  contact 
and  then  centrifugalized. 

B.  In  6  similar  tubes  containing  each  0.5  of  a  cubic  centimeter 
of  a  5  per  cent  suspension  of  bovine  blood  corpuscles  saturated  with 
hemolytic  sensitizer  was  placed  one  drop  of  each  supernatant  fluid 
from  the  tubes  in  "  A"  -series. 

C.  Equal  volumes  of  the  washed  bacilli  from  "A"  series  were 
mixed  with  an  emulsion  of  thrice-washed  frog  leucocytes.    A  com- 
parison was  then  made  of   the  degree  of   phagocytosis  in  "C," 
which    may  begin  at  once,  with  the  degree  of   hemolysis  which 
appears  in  "  B  "  after  considerable  time. 

D.  As  a  control,  6  tubes  were  arranged  containing  each  0.5  of  a 
cubic  centimeter  of  the  same  emulsion  of  sensitized  blood  cor- 
puscles as  in  "  B,"  and  to  each  is  added  one  drop  of  equal  volume 
containing,  respectively,  sV,  ¥o,  sV,   iitf,  ??v  and  5-?^  of  a  cubic 
centimeter    of    fresh    frog    serum    which    was    not     previously 
treated  with  anthrax  bacilli.    The  result  of  this  experiment  is  as 
follows : 

1.  The  intensity  of  phagocytosis  in  "  C  "  decreases  from  tube  1 
to  6.    In  No.  1  the  phagocytosis  is  very  marked,  in  No.  6  none  is 
apparent.     This  is  evident,  since  the  bacilli  fixed  less  opsonin  as 
the  serum  dilution  increased  in  "  A." 

2.  In  "  B,"  hemolysis  appears  in  none  of  the  tubes.    The  reason 
for  this  might  be  that  the  dilution  of  serum  in  "  A  "  was  too  great 
to  show  any  appreciable  alexin  action.     This,  however,  is  not  so, 
because : 

3.  In  "D"  series  there  is  hemolysis  in  the  first  four  tubes,  which 
grows  progressively  less,  and  in  the  last  two  there  is  none.     (In 


392  STUDIES  IN  IMMUNITY. 

these  latter  cases  the  serum  dilution  was  actually  too  great.)     This 
experiment  tells  us,  therefore: 

(a)  That  the  alexin  was  fixed  at  the  same  time  with  the  opsonins ; 
(b)  that  the  intensity  of  the  action  o£  opsonin  and  alexin  decreases 
equally  with  increasing  serum  dilution.  The  parallelism  between 
the  action  of  opsonins  and  alexins  is  therefore  striking. 

I  have  tried  the  reverse  experiment  in  order  to  convince  myself 
whether  a  normal  serum  which  has  previously  been  brought  together 
with  a  suitable  amount  of  sensitized  blood  corpuscles,  in  order  to 
bind  the  complement,  has  lost  its  opsonin  together  with  its  alexin. 
It  might  be  that  the  hemolysis,  which  occurs  after  addition  of  fresh 
alexic  serum  to  sensitized  blood  corpuscles,  might  influence  the 
later  phagocytosis  experiment  with  this  serum  and  check  its 
opsonic  action.  This  consideration  led  me  to  employ  fresh  horse 
serum,  the  alexin  of  which,  as  is  well  known,  becomes  fixed  upon 
sensitized  blood  corpuscles  readily  without  causing  hemolysis. 
Furthermore,  I  had  previously  convinced  myself  that  horse  serum 
exerts  a  pronounced  opsonic  effect  when  added  to  anthrax  bacilli 
and  washed  frog  leucocytes.  The  technic  of  the  experiment  is  as 
follows : 

Tube  A  contains  2  c.c.  of  a  5  per  cent  emulsion  of  bovine  cor- 
puscles (thrice  washed)  and  0.2  of  a  cubic  centimeter  of  inactivated 
specific  hemolytic  serum  (rabbit  >  bovine). 

Tube  B  contains  the  same  amount  of  blood  as  "A"  and  0.2 
of  a  cubic  centimeter  of  physiological  saline  solution. 

The  tubes  are  properly  mixed  and  allowed  to  stand  a  quarter  of 
an  hour.  The  tubes  are  then  filled  up  with  saline  solution,  shaken, 
centrifugalized  and  all  the  supernatant  fluid  carefully  removed 
with  a  pipette.  To  the  sediments  in  the  two  tubes  is  then  added 
0.2  of  a  cubic  centimeter  of  physiological  saline  solution  and  0.1 
of  a  cubic  centimeter  of  fresh  horse  serum.  They  are  shaken  and 
the  mixtures  allowed  to  stand  for  2  hours.  They  are  then  cen- 
trifugalized, and  the  supernatant  liquids  are  employed  for  the  fur- 
ther tests.  It  is  evident  that  if  the  quantities  have  been  properly 
chosen  (and  such  is  the  case  here)  all  the  alexin  of  the  horse  serum 
in  tube  "A"  has  been  bound  by  the  sensitized  blood  corpuscles, 
whereas,  in  tube  "B"  it  is  still  free  in  the  fluid.  As  a  definite  con- 
trol we  may  employ  a  phenomenon  which  has  given  rise  to  a  dispute 


ON  THE  NATURE  OF  OPSONINS. 


393 


between  Ehrlich  and  Sachs*  on  the  one  hand  and  Bordet  and  Gayf 
on  the  other  in  regard  to  the  explanation  of  the  phenomenon  of 
complement  deviation.  We  do  not  wish  here  to  enter  into  this 
theoretical  controversy,  but  merely  mention  the  fact  that  the  bring- 
ing of  diluted  washed  guinea-pig  blood  in  contact  with  inactivated 
bovine  serum  and  fresh  horse  serum  in  suitable  quantities  produces 
rapid  and  very  pronounced  agglutination  (later  hemolysis) ,  for  which 
phenomenon  the  alexin  of  the  horse  serum  is  necessary. 

We  now  set  up  two  tubes  (a  and  b),  each  of  which  contains  0.2  of 
a  cubic  centimeter  of  physiological  saline  solution  and  1  drop  of 
a  1-10  dilution  of  washed  guinea-pig  blood  and  half  a  drop  of  in- 
activated bovine  serum.  To  mixture  "a"  is  added  a  drop  of  the 
liquid  from  the  tube  "A,"  while,  to  the  similar  mixture  "b," 
1  drop  of  the  liquid  from  tube  "B"  is  added.  In  mixture  "b" 
the  blood  corpuscles  are  distinctly  agglutinated  in  about  10  minutes. 
In  "a"  the  agglutination  is  totally  absent  and  remains  so.  Fluid 
"  B  "  contains,  therefore,  free  alexin;  "A,"  however,  does  not. 

Let  us  inquire,  now,  what  the  condition  of  the  opsonic  power  of 
these  two  fluids  is.  For  this  purpose  the  mixtures  indicated  are 
prepared,  using  in  each  case  equal  quantities  of  the  several  com- 
ponents (1  drop  of  each  in  a  hollow  ground  slide  protected  from 
the  air  by  a  cover  glass).  After  a  contact  of  30  minutes  smears 
are  prepared.  The  account  of  one  of  my  experiments  gives  the 
following  results: 


1.  Washed  frog  leucocytes 

An  emulsion  of  anthrax  bacilli 
Physiological  saline 

2.  Washed  frog  leucocytes 
Emulsion  of  anthrax  bacilli 
Fluid  from  tube  "A" 

3.  Leucocytes 
Anthrax  bacilli 

Fluid  from  tube  "B" 

4.  Leucocytes 

Emulsion  of  typhoid  bacilli 
Physiological  saline  solution 

5.  Leucocytes 

Emulsion  of  typhoid  bacilli 
Fluid  "A" 

6.  Leucocytes 

Emulsion  of  typhoid  bacilli 
Fluid  "B" 


Result,  no  phagocytosis. 

28  per  cent  of  the  leucocytes  counted 
engaged  in  active  phagocytosis. 

78  per  cent  of  the  leucocytes  show 
phagocytosis. 

Minimal  phagocytosis. 

19  per  cent  of  the  counted  leucocytes 
are  more  or  less  filled  with  typhoid 
bacilli. 

83  per  cent  of  the  leucocytes  show 
phagocytosis. 


*  Studies  on  Immunity,  Ehrlich-Bolduan,  John  Wiley  and  Sons,  p.  209. 
t  See  p.  363. 


394  STUDIES  IN   IMMUNITY. 

This  experiment  shows,  therefore,  that  the  opsonin  disappear 
to  a  great  extent  from  the  serum  together  with  the  alexin. 

I  should  like  to  cite  a  final  experiment  upon  the  binding  or  fix- 
ation of  alexin  in  support  of  the  conception  of  the  identity  of  normal 
opsonin  and  alexin.  It  is  well  known  that  rabbit  blood  is  strongly 
hemolyzed  by  fresh  dog  serum.  If  suitable  doses  are  used, the  rabbit 
blood  corpuscles  will  fix  all  the  normal  sensitizers  and  all  the  alexin 
of  the  dog  serum.  This  fact  is  made  use  of  in  the  following  experi- 
ment: 

Tube  1.  0.2  of  a  cubic  centimeter  of  normal  defibrinated  rabbit 
blood  is  washed  three  times  with  physiological  saline  solution. 
After  the  third  washing  all  the  supernatant  fluid  is  removed  with  a 
pipette.  To  the  centrifugalized  blood  corpuscles  is  added  0.2  of  a 
cubic  centimeter  of  fresh  dog  serum  and  2  drops  physiological 
saline  solution.  They  are  properly  mixed  and  allowed  to  remain 
in  contact  2  hours. 

Tube  2.  0.2  of  a  cubic  centimeter  of  a  thick  emulsion  of  anthrax 
bacilli  (24-hour  agar  culture)  plus  0.2  of  a  cubic  centimeter  of 
fresh  dog  serum  and  2  drops  of  physiological  saline  solution, 
mixed,  and  left  in  contact  2  hours.  After  2  hours  both  tubes  are 
centrifugalized.  In  tube  No.  1  a  marked  hemolysis  has  taken 
place.  As  a  control  to  determine  the  content  of  alexin  in  the  fluid 
of  tube  2,  the  following  three  tubes  are  made: 

A.  0.5  of  a  cubic  centimeter  washed  rabbit  blood ;  1  drop  of 
fresh  dog  serum. 

B.  0.5  of  a  cubic  centimeter  washed  rabbit  blood;  1  drop  of 
fluid  from  tube  2. 

C.  0.5  of  a  cubic  centimeter  washed  rabbit  blood;  1  drop  of 
physiological  saline  solution. 

Contact,  2  hours. 
Result:  A.    Hemolysis. 

B.  Faint  hemolysis,  distinctly  less  than  in  A. 

C.  No  hemolysis. 

The  hemolysis  in  "A"  is  naturally  less  than  in  tube  1  because  in 
the  latter  tube  equal  parts  of  blood  and  serum  had  been  brought 
together  and  the  dilution  of  the  serum  in  "A"  is  therefore  much 
greater.  The  important  point  is  that  the  weaker  hemolysis  in  "  B  " 
proves  that  in  tube  2  a  fixation  of  alexin  on  the  bacteria  has  taken 


ON  THE  NATURE  OF  OPSONINS.  395 

place.  The  control  with  the  fluid  from  tube  1  can,  of  course,  not  be 
made  up  because  hemoglobin  has  already  been  dissolved  there. 
The  following  opsonin  experiments  were  furthermore  carried  out: 

1 .  Washed  dog  leucocytes 

Anthrax  bacilli  [     Very  marked  phagocytosis. 

Fresh  dog  serum 

2.  Leucocytes  1 

Anthrax  bacilli  >     Weak  phagocytosis. 

Fluid  from  tube  1 

3.  Leucocytes  1 

Anthrax  bacilli  [     Slight  phagocytosis. 

Fluid  from  tube  2 

4.  Leucocytes  as  in  1,  2  and  3 
Bacilli  from  tube  2  washed  once 


No  serum;  merely  some  saline 
solution. 


Very  pronounced  phagocytosis. 


The  opsonin  has,  therefore,  in  great  part  disappeared  from  the 
dog  serum  (2  and  3),  together  with  the  alexin,  having  become  fixed 
in  tube  1  to  the  blood  corpuscles  (hemolysis)  and  in  tube  2  to  the 
bacteria  (4). 

All  these  facts  lead  us  to  the  conviction  that  in  the  normal  serum 
the  parallelism  between  the  action  of  the  alexin  and  the  opsonin 
goes  so  far  that  they  can  no  longer  be  considered  as  different,  but 
must  be  regarded  as  identical.  This  conclusion  agrees  very  well 
with  the  frequently  proven  fact  which  has  also  been  shown  by  me 
to  hold  for  frog  serum,  namely,  that  normal  opsonin  is  thermolabile. 

I  *  have  previously  pointed  out  the  difference  between  the  op- 
sonin and  the  agglutinin  in  frog  serum  for  anthrax  bacilli  in  that 
they  are  destroyed  at  different  temperatures  (opsonin  at  56°,  agglu- 
tinin at  70°).  A  further  proof  is  found  in  tubes  5  and  6  of  the 
series  on  page  393.  The  differences  in  the  degree  of  phagocytosis 
are  particularly  large,  here,  whereas  the  extracellular  typhoid 
bacilli  in  the  preparations  of  both  tubes  were  slightly  agglutinated 
by  the  horse  serum. 

In  investigating  the  opsonins  of  specific  sera  I  have  employed 
hemolytic  sera.  Savtchenko  f  has  previously  shown  that  blood 
corpuscles  that  are  loaded  with  specific  hemolytic  sensitizers  easily 
fall  prey  to  the  leucocytes 4 

*  Loc.  cit. 

f  Annales  de  1'Institut  Pasteur,  December,  1907. 

J  One  must  remember  that  these  experiments  were  performed  with  unwashed 
exudate  leucocytes,  and  other  influences  cannot  be  excluded, 


396  STUDIES  IN   IMMUNITY. 

Metchnikoff  *  himself  has  accepted  this  view  and  ascribed  to  the 
fixateur  in  general  an  adjuvant  role  in  phagocytosis.  Later,  after 
Wright's  investigations  had  turned  the  discussion  to  the  opsonin, 
there  were  endeavors  made  to  demonstrate  opsonins  for  blood 
corpuscles  in  specific  hemolytic  sera.  Let  us  see  if  this  was  justi- 
fiable. In  experiments  with  an  exudate  of  macrophages  from  a 
rabbit  I  found  that  the  washed  leucocytes  took  up  very  actively 
bovine  blood  corpuscles  which  had  first  been  treated  with  specific 
hemolytic  sensitizer  (inactivated  rabbit  >  bovine  serum)  and 
then  with  alexin  (fresh  horse  serum) ,  but  that  only  sensitized,  not 
alexinized,  blood  corpuscles  were  taken  up  by  the  leucocytes  in 
less  degree.  Washed  untreated  blood  corpuscles  were  not  taken 
up  by  phagocytes.  I  repeated  these  observations  with  thrice- 
washed  dog  leucocytes  in  the  following  manner: 

1.  Leucocytes. 

Washed  bovine  corpuscles. 

2.  Leucocytes. 

Sensitized  bovine  corpuscles. 

3.  Leucocytes. 

Sensitized  and  alexinized  bovine  corpuscles. 

4.  Leucocytes. 

Bovine  corpuscles  plus  alexin. 

Contact,  1  hour  at  37  degrees.  The  blood  of  2  and  3  was  washed 
after  each  preparatory  treatment  with  salt  solution,  whereby  only 
those  constituents  of  the  serum  which  the  corpuscles  had  fixed 
could  develop  any  action,  and  all  else  was  removed.  The  experi- 
ment gave  the  result  that  in  1  and  4  the  phagocytosis  is  about  nul. 
In  2  it  is  fairly  strong,  whereas  in  3  there  is  hardly  a  macro-  or 
microphage  which  does  not  contain  one  or  more  blood  corpuscles. 
The  free  alexin  (4)  is  not  able,  therefore,  alone  to  stimulate  phago- 
cytosis, whereas  it  is  able  when  the  specific  sensitizers  are  fixed  to 
blood  corpuscles  to  increase  (3)  their  already  well-marked  action  (2). 

Similar  experiments  with  another  hemolytic  serum  (rabbit  > 
goat  and  goat  corpuscles)  gave  entirely  corresponding  results. 

I  have  not  yet  observed  a  specific  serum  which  was  hemolytic 
but  not  hemotropic,  as  reported  by  Neufeld  and  Topfer.f  Our 
results,  however,  show  a  distinct  similarity  with  the  findings  of 

*  Han<ll).  d«-r  Pathog.,  Microorg.,  Bd.  IV,  Teil.  I. 
t  Cent,  fiir  Bakt.,  Bd.  XXXVIII,  p.  456. 


ON  THE  NATURE  OF  OPSONINS.  397 

Levaditi*  on  bacterial  sera.  We  have  then  in  our  hemolytic  sera 
also  a  thermolabile  opsonin  which  becomes  fixed  to  the  blood  cells 
and  thus  causes  phagocytosis.  It  is  at  once  suggested  that  here 
the  opsonin  may  be  regarded  as  identical  with  the  specific  sensi- 
tizer.  One  can,  as  a  matter  of  fact,  accurately  remove  the  specific 
sensitizer  from  a  hemolytic  serum  by  previously  treating  it  with 
the  corresponding  blood  corpuscles  and  then  prove  that  it  has  at 
the  same  time  and  in  the  same  degree  lost  its  opsonic  power.  Thus 
Wakelin  Barrattf  in  his  experiments  on  the  quantitative  opsonic 
absorption  by  blood  corpuscles  from  an  inactivated  hemolytic 
serum  worked  only  with  the  specific  sensitizers.  If  one  adds  a 
fresh  specific  serum  to  blood  corpuscles  or  bacteria,  a  combined 
lytic  and  opsonic  action  by  sensitizers  and  alexin  is  obtained,  as  in 
tubes  2  and  3  of  the  last  experiment. 

Let  us  return  for  a  moment  to  our  experiment  with  normal  serum. 
We  have  seen  that  the  opsonin  was  fixed  by  specific  sensitizers 
with  the  alexin  and  was  also  destroyed  by  heat.  There  remain, 
however,  in  the  normal  sera  after  fixation  or  destruction  of  the 
opsonins  (alexins)  almost  constantly  a  few  opsonic  effects  (compare 
page  363,  tubes  2  and  5)  which  are  much  greater  in  the  specific  sera. 
And  since  we  do  not  find  any  reason  for  assuming  that  the  alexin 
in  the  normal  serum  becomes  fixed  to  the  bacteria  without  the  in- 
tervention of  sensitizers  we  have  to  assume,  that  the  opsonic 
action  either  of  normal  or  of  specific  sera  is  in  the  main  part 
a  combined  action  of  sensitizers  and  alexin.  In  normal  serum 
it  is  caused  chiefly  by  the  alexin.  In  the  specific  serum  it  is 
increased  and  brought  about  chiefly  by  the  sensitizers.  I  concur, 
therefore,  with  DeanJ  in  the  belief  that  normal  as  well  as  specific 
opsonins  have  a  dualistic  complexity.  § 

*  C.  R.  Soc.  de  Biologie,  April  and  May,  1907. 
t  Proc.  Roy.  Soc.,  1905  and  1907. 
j  Proc.  Roy.  Soc.,  1905  and  1907. 

§  See,  further,  my  dissertation,  "Phagocytose  en  Opsoninen,"  Amsterdam, 
1908,  where  I  explain  more  fully  the  nature  and  action  of  the  opsonins. 


XXL    ALEXIN  ABSORPTION   AND  THE   ANTAGONISTIC 
PROPERTY  OF  NORMAL  SERA.* 

BY    JULES   BORDET  AND   FREDERICK  P.   GAY. 

The  alexin  fixation  method  has  been  used  for  many  purposes. 
The  method  depends  on  the  factf  that  bacteria  or  red  blood  cells 
when  treated  with  a  suitable  sensitizer  acquire  the  property  of  fixing 
alexin,  and  of  removing  it  from  the  surrounding  fluid.  By  this 
method  it  was  shown  seven  years  agoj  that: 

(a)  Animals  vaccinated  against  bacteria  usually  produce  specific 
sensitizers,  whatever  be  the  organism  employed. 

(6)  And,  consequently,  although  many  bacteria  resist  bacterioly- 
sis by  specific  immune  serum  it  is  not  owing  to  a  lack  of  active 
substances  in  the  serum,  but  on  account  of  resistance  on  the  part 
of  the  bacteria. 

(c)  Fixation  of  alexin  in  the  presence  of  immune  serum  may  be 
used  as  a  means  of  diagnosing  bacteria  and  is  of  even  more  general 
applicability  than  is  agglutination,  for  a  sensitizing  action  is 
often  present  when  agglutination  is  absent. 

A  year  later  Gengou§  demonstrated  that  sensitizers  appear  after 
immunization  with  substances  other  than  bacteria  and  corpuscles; 
he  found  that  the  serum  of  an  animal  immunized  against  an  amor- 
phous albuminous  substance  (casein,  fibrinogen,  alien  serum,  etc.) 
sensitizes  the  specific  substance  in  question,  and  so  endows  it  with 
the  property  of  absorbing  alexin. 

The  subsequent  applications  of  this  method  and  the  technic 
employed  are  too  well  known  to  require  mention.  The  results 
obtained,  however,  may  be  considerably  affected  by  certain  factors 

*  L'absorption  de  1'alexine  et  le  pouvoir  antagoniste  des  scrums  normaux. 
Annales  de  1'Institut  Pasteur,  XXII,  1908,  625. 
t  Bordet,  Hemolytic  sera,  etc.,  p.  186. 

I  Bordet  and  Gengou,  The  existence  of  sensitizers  etc.,  p.  217. 
$  Gengou,  On  the  sensitizers  of  sera  active  against  albuminous  substances, 

p    241. 

398 


ALEXIN  ABSORPTION.  399 

that  have  not  always  been  as  carefully  considered  as  they  should 
have  been.  One  of  these  influences,  which  we  wish  to  consider  in 
particular,  is  the  amount  of  normal  salt  solution  that  is  used  in  the 
mixtures.  Variations  in  this  factor  are  evident  in  many  experi- 
ments on  hemolysis,  and  such  variations,  we  believe,  explain  certain 
phenomena  that  have  been  noted  by  Pfeiffer  and  Friedberger  and 
by  Sachs,  to  which  they  have  referred  as  the  antagonistic  property 
of  normal  sera.  Sachs  offers  a  rather  complicated  explanation  of 
this  property,  which,  as  we  shall  later  see,  appears  to  us  incorrect. 

When  fresh  normal  serum  (alexin)  is  mixed  with  a  suitable  sen- 
sitizing serum  (heated  to  55  degrees)  and  the  substance  under 
consideration,  usually  red  blood  cells  or  bacteria,  to  determine  alexin 
fixation,  the  cells  employed  are  usually  added  as  a  suspension  in 
salt  solution.  The  amount  of  this  solution,  however,  varies;  in 
dealing  with  red  blood  corpuscles  many  workers  use  a  considerable 
dilution,  for  example,  a  5  per  cent  suspension,  and  a  relatively  large 
volume  of  this  suspension  is  therefore  employed. 

It  has  frequently  been  noted  by  experimenters  that  hemolysis 
in  salt  solution  is  very  rapid.  A  given  quantity  of  corpuscles  is 
generally  hemolyzed  much  more  rapidly  by  a  given  dose  of  alexin 
and  sensitizer  when  the  liquid  that  serves  as  a  medium  is  salt 
solution  than  when  it  is  normal,  and  supposedly  inert,  heated 
serum.*  On  comparing  normal  inert  serum  with  salt  solution,  we 
find  that  the  latter  favors  hemolysis,  or,  more  correctly  speaking, 
that  the  normal  serum  inhibits  hemolysis;  it  is  to  be  noted  that 
this  difference  is  very  marked  only  when  the  corpuscles  are  not 
strongly  sensitized.  To  what  is  the  antagonistic  effect  due?  As 
both  Pfeiffer  and  Friedberger  (bacteriolysis)  and  Sachs  have  shown, 
the  normal  serum  does  not  act  by  weakening  the  sensitization ;  nor 
directly  on  the  corpuscles  or  the  bacteria.  We  shall  presently 
consider  Sachs'  explanation,  which  supposes  that  the  serum  owes 
its  inhibiting  effect  to  the  presence  of  normal  amboceptors  that 

*  Muller  (Central,  fur  Bakt.,  XXX,  1901)  was,  we  believe,  the  first  to  note  this 
fact  in  particular.  It  has  been  more  recently  considered  by  other  investigators, 
and  in  particular  by  Muir  and  Browning  (Jour,  of  Hygiene,  VI,  1906),  and  by 
Liebermann  and  Fenivessy  (Peters.  Me"dic.  Chirug.  Pressen,  1907),  etc.  We  have 
already  mentioned  (see  p.  383.)  Klein's  researches  and  our  own  on  horse  serum, 
in  which  it  was  shown  that  alexin  is  better  absorbed  by  corpuscles  when  salt 
solution  is  present. 


400  STUDIES   IN   IMMUNITY. 

monopolize  the  alexin.  As  Muir  and  Browning  have  correctly 
stated,  the  serum  is  antagonistic  because  it  prevents  the  fixation  of 
the  alexin  on  the  sensitized  cells,  and  this  antagonism  is  overcome 
only  when  the  avidity  of  the  cells  for  the  alexin  is  very  marked, 
namely,  when  the  cells  are  strongly  sensitized  or  when  the  absorp- 
tion of  alexin  is  favored  by  the  addition  of  salt  solution. 

Sachs'  experiment,  which  deals  with  the  antagonistic  action  of 
normal  rabbit  serum  against  the  hemolysis  of  bovine  corpuscles 
treated  with  a  small  amount  of  sensitizer  (the  serum  of  a  rabbit 
immunized  against  bovine  blood)  in  the  presence  of  alexin  (fresh 
guinea-pig  serum)  is  a  suitable  one  for  the  study  that  concerns  us. 
It  will  be  well  to  consider  our  experiment  somewhat  in  detail  in 
order  to  deal  with  Sachs'  explanation  of  a  similar  experiment  in 
which  normal  rabbit  serum  was  also  employed.*  The  following 
experiment  shows  that  moderately  sensitized  corpuscles  are  hemo- 
lyzed  by  alexin  if  the  medium  in  which  they  are  suspended  contains 
a  relatively  large  amount  of  salt  solution,  whereas  they  remain  intact 
or  are  affected  only  very  slowly,  if  the  surrounding  fluid  contains 
less  salt  solution  and  a  correspondingly  increased  amount  of  normal 
rabbit  serum  (heated  to  56  degrees). 

Tube  A  contains  0.6  of  a  cubic  centimeter  of  physiological  solu- 
tion (NaCl,  0.9  per  cent) ;  Tube  B,  0.3  of  a  cubic  centimeter  of  salt 
solution  plus  0.3  of  a  cubic  centimeter  of  normal  rabbit  serum 
(56  degrees  for  one-half  hour).  To  each  tube  is  added  0.05  of  a 
cubic  centimeter  of  alexin  (fresh  normal  guinea-pig  serum)  and  0.3 
of  a  cubic  centimeter  of  salt  solution  containing  10  per  cent  of 
moderately  sensitized  bovine  blood. f 

Tubes  A  and  B  are  placed  in  a  thermostat  (35  degrees).     Hemoly- 

*  Rabbit  serum  is  used,  as  it  is  only  faintly  sensitizing  for  ox  corpuscles.  It  is 
evident  that  if  the  normal  serum  has  a  distinct  sensitizing  effect  on  the  corpuscles 
under  consideration  it  cannot  be  considered  as  an  inert  medium.  Theoretically 
there  is  perhaps  no  absolutely  inert  serum,  but  its  sensitizing  effect  on  a  given 
bacterium  or  alien  red  blood  cell  is  frequently  so  slight  as  to  be  imperceptible,  as 
is  the  case  in  the  present  instance. 

t  This  blood  is  prepared  as  follows:  To  1  c.c.  of  bovine  blood  (previously 
washed  to  remove  all  serum)  is  added  9  c.c.  of  salt  solution,  and  0.1  of  a  cubic 
centimeter  of  serum  (56  degrees)  from  a  rabbit  immunized  against  bovine  blood. 
After  shaking  and  allowing  contact  for  20  minutes  the  tube  is  centrifugalized  and 
the  supernatant  fluid  removed.  To  the  sediment  of  corpuscles  10  c.c.  of  salt 
solution  is  then  added. 


ALEXIN  ABSORPTION.  401 

sis  is  complete  in  A  in  half  an  hour.  The  corpuscles  in  B  are 
intact  after  3  hours;  slight  hemolysis  subsequently  takes  place. 
It  is  scarcely  necessary  to  mention  that  control  tubes  A  T  and 
B  T,  identical  with  A  and  B  respectively,  but  containing  non- 
sensitized  bovine  blood,  are  likewise  prepared;  in  these  mixtures 
there  is  no  hemolysis. 

It  is  evident,  then,  that  a  small  amount  of  normal  serum  inhibits 
hemolysis  distinctly;  further  experiments  show  that  this  antago- 
nistic effect  varies  in  intensity  with  the  increase  of  heated  normal 
serum  and  the  decrease  of  salt  solution. 

The  question  arises  as  to  how  intense  this  antagonistic  power  in 
normal  serum  is.  We  can  also  determine  whether  normal  serum 
combats  one  or  both  the  factors  of  hemolysis  (alexin  and  sensitizer). 
We  find  that  the  serum  shows  a  relatively  retarding  effect  on  alexin 
even  if  the  dose  of  it  is  relatively  high.  For  example: 

Mixture  A  contains :  0.6  c.c.  of  salt  solution,*  0.05  c.c.  guinea-pig 
alexin,  and  0.2  c.c.  of  salt  solution  containing  40  per  cent  of  sen- 
sitized bovine  blood. t  Mixture  B  contains  the  same  amount 
of  blood  and  twice  as  much  alexin,  but  0.6  c.c.  of  normal  rabbit 
serum,  56  degrees,  in  place  of  the  salt  solution.  Hemolysis  is  more 
rapid  in  A  than  in  B. 

When  corpuscles  are  heavily  sensitized,  however,  the  antihemo- 
lytic  effect  of  normal  serum  (56  degrees)  is  less  evident,  although 
still  distinct.  In  the  following  experiment  even  small  doses  of 
serum  show  distinct  effects: 

Tubes  A  and  C.     Each  0.7  c.c.  of  salt  solution. 

Tubes  B  and  D.  Each  0.4  c.c.  of  salt  solution  plus  0.3  c.c.  of 
heated  normal  rabbit  serum. 

To  A  and  B  is  then  added  0.25  c.c.  of  salt  solution  containing 
40  per  cent  faintly  sensitized  ox  blood  (1  part  of  rabbit  anti- 
bovine  sensitizer  to  10  parts  of  blood) ;  to  tubes  C  and  D  is  added 
the  same  amount  of  blood  sensitized  with  three  times  as  much 
sensitizer.  To  each  of  the  four  tubes  is  then  added  0.05  c.c.  of 
guinea-pig  alexin  and  the  tubes  are  placed  at  35  degrees.  Hemoly- 

*  In  these  experiments  0.9  per  cent  NaCl  is  used. 

f  This  blood  is  relatively  well  sensitized  and  is  prepared  as  follows:  To  1  c.c. 
of  washed  blood  is  added  9  c.c.  of  salt  solution  and  0.5  c.c.  of  rabbit-antibovine 
serum,  56  degrees.  Contact,  1  hour.  Centrifugalization  and  removal  of  the 
supernatant  fluid.  2.5  c.c.  of  salt  solution  is  added  to  the  sediment. 


402  STUDIES  IN   IMMUNITY. 

sis  is  complete  in  C  in  12  minutes,  and  in  A  in  35  minutes.    In  D 
hemolysis  is  complete  in  1  hour.     B  is  not  hemolyzed. 

To  what  is  the  inhibiting  effect  of  normal  serum  due?  It  may 
easily  be  shown  that  the  serum  does  not  act  by  suppressing  the  sen- 
sitization  of  the  corpuscles.  This  has  been  demonstrated  by  our 
predecessors  by  the  following  simple  experiment :  In  a  mixture  of 
moderately  sensitized  ox  corpuscles,  heated  normal  serum  and 
guinea-pig  alexin  there  is  no  hemolysis  owing  to  the  antagonistic 
power  of  the  normal  serum.  On  subsequent  centrifugalization  and 
decantation,  however,  the  addition  of  salt  solution  containing  a 
trace  of  alexin  causes  hemolysis,  which  shows  that  the  corpuscles 
have  retained  their  sensitization. 

This  result  also  shows  that  heated  normal  serum  does  not  directly 
render  the  corpuscles  refractory  to  hemolysis.  One  might  suppose 
that  such  a  serum  contains  "  complementoids "  that  satisfy  the 
affinities  of  the  sensitized  corpuscle  and  hinder  the  subsequent 
fixation  of  active  alexin.  The  preceding  experiment  would,  how- 
ever, invalidate  this  supposition. 

The  normal  serum,  in  addition  to  having  no  effect  on  the  sensiti- 
zation of  the  corpuscle,  has  also  no  neutralizing  effect  on  the  alexin. 
It  does,  however,  oppose  the  fixation  of  the  alexin  on  the  sensitized 
corpuscles,  and  this  explains  the  lack  of  hemolysis. 

The  proof  that  normal  serum  does  not  alter  the  alexin  is  given  by 
the  fact  that  hemolysis  can  be  produced  in  an  inhibited  mixture  of  nor- 
mal serum,  alexin  and  sensitized  corpuscles  by  adding  salt  solution; 
in  other  words,  as  already  stated,  the  opposition  to  hemolysis  depends 
on  the  concentration  of  normal  serum.  Muir  and  Browning  in  study- 
ing this  fact  have  clearly  shown  that  the  alexin  is  not  fixed  on  the 
corpuscles  during  the  first  phase  of  the  experiment,  when  the  mixture 
contains  much  serum  and  little  salt  solution,  but  is  absorbed  only 
on  the  addition  of  the  saline  solution.  Similar  results  are  obtained 
if,  as  we  have  performed  the  experiment,  corpuscles  before  dilution 
in  salt  solution  are  sufficiently  sensitized  to  produce  their  dissolu- 
tion. Such  an  hemolysis,  to  be  sure,  naturally  brings  about  an 
absorption  of  alexin,  which,  however,  is  only  partial  if  suitable 
amounts  are  used  and  the  sensitization  is  not  too  great;  such  ab- 
sorption always  remains  less  than  when  salt  solution  is  added. 

*** 


ALEXIN  ABSORPTION.  403 

What  ideas  concerning  the  mode  of  union  of  the  alexin  with  sen- 
sitized corpuscles  are  suggested  by  these  facts?  One  of  us*  has 
already  expressed  the  opinion  that  this  fixation  of  alexin  is  not  a 
chemical  combination,  strictly  speaking,  between  the  alexin  and  a 
particular  group  of  the  sensitizer  (complementophilic  group  of  the 
amboceptor,  according  to  Ehrlich),  but  represents  simply  a  phenom- 
enon of  molecular  adhesion  (adsorption).  This  point  of  view  was 
still  further  elaborated  in  1906f  by  the  authors  of  the  present 
article.  We  regard  the  union  of  the  sensitizer  and  red  blood  cells  as 
constituting  a  complex  with  more  adsorption  avidity  for  the  alexin 
than  the  normal  corpuscle :  the  alexin  tends  to  precipitate  on  the 
sensitized  corpuscle,  the  alteration  of  which  is  the  more  marked 
the  greater  the  sensitization.  How,  from  this  standpoint,  does 
the  inhibiting  serum  act?  It  would  seem  to  us  that  it  holds  the 
alexin  in  a  state  of  more  or  less  definite  suspension  in  the  medium, 
and  gives  it  a  more  stable  equilibrium;  with  salt  solution,  on  the 
other  hand,  the  equilibrium  is  more  unstable,  that  is  to  say,  the 
alexin  condenses  or  precipitates  more  readily  on  those  cells  that 
attract  it. 

This  idea  seems  to  us  to  be  in  harmony  with  the  observations 
of  Gengou  on  colloids  and  suspensions  of  inorganic  precipitates. 
GengouJ  found  that  if  washed  red  blood  corpuscles  are  added  to 
salt  solution  containing  such  an  inert  inorganic  precipitate  as  barium 
sulphate  in  suspension,  the  cells  and  the  precipitate  clump  and 
hemolysis  occurs.  But  if  a  trace  of  serum  is  previously  added 
to  the  precipitate,  this  phenomenon  of  agglutination  and  hemolysis 
does  not  occur.  It  was  found  that  serum  causes  a  dissociation  of 
the  particles  of  barium  sulphate  and  gives  it  a  milky  appearance 
which  delays  the  clarification  of  the  fluid  by  sedimentation.  And 
serum,  then,  inhibits  the  precipitation  of  alexin  on  the  corpuscles 
just  as  it  prevents  the  sedimentation  of  barium  sulphate. 

Citrate  of  sodium  produces  an  effect  on  barium  sulphate  similar 
to  the  one  caused  by  serum,  and  also  changes  it  into  a  milky  fluid 
and,  as  Gengou  has  found,  deprives  it  of  its  property  of  agglutinat- 
ing and  hemolyzing  red  blood  cells.  And  there  is  a  still  further 
analogy,  in  respect  to  the  citrate,  between  the  precipitation  of 

*  Bordet,  Hemolytic  sera,  p.  186,  and  Cytolytic  sera,  p.  228. 
t  Bordet  and  Gay,  On  the  relation  of  sensitizers,  etc.,  p.  363. 
J  Gengou,  Researches  on  the  agglutination  of  red  blood  cells,  etc.,  p.  312. 


404  STUDIES  IN  IMMUNITY. 

barium  sulphate  on  corpuscles,  and  the  fixation  of  alexin  on  sensi- 
tized cells.  We  have  found  that  sodium  citrate  in  suitable  doses 
will  protect  red  blood  cells  from  hemolytic  sera,  and  it  may  further 
be  shown  that  it  acts  by  preventing  the  fixation  of  the  alexin  on 
the  sensitized  cells.*  The  analogy  between  barium  sulphate  and 
alexin  is  evidently  suggestive. 

The  following  experiment  may  be  cited  as  offering  an  adequate 
though  somewhat  homely  comparison  as  to  the  mode  of  union  of 
the  alexin  with  sensitized  corpuscles:  Water  rolls  over  the  surface 
of  a  watch  glass  covered  with  paraffin  without  adhering  to  it.  If 
the  water  contains  barium  sulphate  in  suspension,  the  paraffin 
becomes  wet  in  a  few  minutes  and  the  drop  spreads  out  over  the 
surface,  owing  to  the  fact  that  the  surface  is  covered,  by  molecular 
adhesion,  with  a  thin  white  stratum  of  barium  sulphate.  This 
stratum  is  wet  by  water  and  will  even  resist  rinsing,  being  remov- 
able only  by  friction.  But  if  we  use  intead  a  suspension  of  barium 
sulphate  that  has  been  rendered  milky  by.  citrate,  no  such  phen- 
omenon occurs;  under  such  conditions  the  sulphate  fails  to 
cover  the  paraffin  and  the  surface  does  not  wet.  In  other  words, 
the  citrate  prevents  the  precipitation  of  the  sulphate  on  paraffin 
just  as  it  does  the  precipitation  of  alexin  on  sensitized  corpuscles. 

We  shall  not  consider  the  mechanism  of  this  phenomenon  at 
this  point  nor  insist  on  the  properties  of  the  citrate  in  hemolysis, 
as  these  subjects  have  already  been  studied  in  detail  by  Gengouf 
in  our  Institute. 

There  are  certain  pertinent  considerations  that  occur  in  this 
connection  applicable  to  the  methods  based  on  absorption  of  alexin, 
and  in  a  general  way  of  service  in  studies  on  hemolysis  and  bacte- 
riolysis. 

In  the  first  place  the  facilitating  effect  of  a  large  amount  of  salt 
solution  on  alexin  fixation  should  never  be  lost  sight  of.  The 

*  After  proving  that  sensitized  blood  corpuscles  remain  intact  in  a  mixture 
of  citrate  and  alexin,  it  may  subsequently  be  demonstrated,  after  centrifugal  i/a- 
tion,  that  the  supernatant  fluid  still  contains  alexin  and  is  able  to  hemolyze  new 
sensitized  corpuscles;  for  this  purpose  a  little  calcium  chloride  is  added  to  neu- 
tralize the  citrate.  Control  tubes  show  that  a  fixation  of  alexin  does  take  place 
when  no  citrate  is  added. 

t  Several  observations,  particularly  as  regards  hemolysis  by  eel  serum  and  by 
venom,  have  been  published  by  Gengou  in  the  Bulletin  de  la  Socidte"  de  Biologic 
(1907).  See  also  p.  414. 


ALEXIN  ABSORPTION.  405 

experimental  method  employed  to  demonstrate  alexin  fixation 
consists,  as  we  know,  of  two  phases:  In  the  first  phase  the  alexin 
is  placed  in  contact  with  such  cells  as  bacteria  plus  a  serum  "A" 
that  is  capable  (or  supposedly  capable)  of  sensitizing  these  cells, 
that  is  to  say,  of  conferring  on  them  the  property  of  fixing  alexin. 
In  the  second  phase  one  determines,  by  adding  sensitized  blood 
corpuscles,  whether  the  alexin  has  disappeared  from  the  fluid  or 
remained  free  in  it ;  in  the  latter  instance  the  fixation  takes  place,  not 
during  the  first  phase  (on  the  bacteria),  but  during  the  second  phase 
(on  the  corpuscles).  As  a  result,  hemolysis  occurs,  and  the  conclu- 
sion is  that  serum  "A"  is  not  sensitizing,  or,  at  best,  only  slightly  so. 
But  it  is  evident  that,  for  experimental  accuracy,  the  two  phases  of 
the  experiment  should  be  comparable  in  respect  to  the  facility  for 
alexin  absorption.  As  we  have  seen,  an  excess  of  serum  inhibits 
fixation  and  an  excess  of  salt  solution  favors  it;  the  effort,  then, 
should  be  to  maintain  a  constant  proportion  between  salt  solution 
and  serum  during  the  entire  experiment.  The  sensitized  corpuscles 
finally  introduced  are  suspended  in  salt  solution;  it  is  evidently 
better  to  add  the  number  of  corpuscles  desired,  not  by  employing 
a  large  volume  (lc.c.,for  example)  of  a  weak  suspension  of  corpuscles, 
but  a  small  volume  of  a  thick  suspension  (0.1  c.c.,  for  example). 
If,  for  example,  the  first  mixture  is:  alexin,  0.1  c.c.,  plus  bacterial 
suspension  0.3  c.c.,  and  0.3  or  0.5  c.c  of  the  serum  in  which  the  pres- 
ence of  a  bacterial  sensitizer  is  to  be  determined,  the  fixation  of  alexin 
on  the  bacteria  will  be  opposed  by  the  large  proportion  of  serum 
in  the  mixture  and  may  be  complete  only  in  case  the  sensitizer  is 
very  strong.  If  much  salt  solution  is  added  with  the  red  blood 
corpuscles,  the  antagonistic  effect  is  removed  and  a  most  minute 
trace  of  free  alexin  will  produce  hemolysis.  And  as  the  antimicrobial 
sensitizer  has  been  handicapped  by  the  hemolytic  sensitizer  it  may 
easily  escape  detection.  Or,  indeed  (and  this  error  would  seem  to  us 
to  have  been  committed),*  one  might  conclude  that  the  alexin  fixed 
by  a  given  cell  (bacterium,  for  instance)  is  not  identical  with  the  one 
fixed  by  a  different  cell  (corpuscle),  and  might  be  led  to  agree  with 
the  erroneous  hypothesis  that  the  bacteriolytic  alexindiffers  from  the 

*  We  think  that  certain  experiments  of  Moreschi  that  seem  to  indicate  a 
plurality  of  alexins  are  open  to  criticism  from  this  standpoint.  (Berlin  klin. 
Wochen.,  1907,  1206);  this  investigator  usually  employs  a  large  volume  of  salt 
solution  in  which  to  suspend  his  sensitized  corpuscles  (1  c.c.).  - 


406  STUDIES   IN   IMMUNITY. 

hemolytic  alexin.  The  numerous  and  varied  applications  of  this 
method  invariably  show  that  the  corpuscles  used  as  an  indicator  of 
alexin  fixation  remain  intact,  whatever  be  the  sensitized  cells  em- 
ployed in  the  first  phase  of  the  experiment,  provided  the  sensitizer  em- 
ployed is  sufficiently  strong  and  the  precautions  mentioned  are  taken 
in  regard  to  the  volume  of  salt  solution.  In  a  corresponding  manner 
account  should  be  taken  of  the  respective  potency  of  the  two  sen- 
sitizers  which  are  successively  used,  in  respect  to  the  fixation  of  a 
given  alexin.  If  the  first  is  not  very  powerful  and  does  not  remove 
the  last  traces  of  alexin,  the  met  hod  may  give  very  varying  results,  de- 
pend ing  on  whether  the  corpuscles  used  as  an  indicator  are  moderately 
or  strongly  sensitized,  for  the  traces  of  alexin  may  be  sufficient  to 
produce  hemolysis  in  the  latter  case,  whereas  in  the  former  instance, 
owing  to  an  antagonistic  effect  of  the  serum  which  tends  to  dis- 
seminate the  alexin,  they  may  not.  Two  opposing  forces  of  vari- 
able energy  tend  to  take  hold  of  the  alexin  and  the  result  depends 
on  the  equilibrium  established  between  them.  With  this  idea  in 
mind  it  is  easy  to  show  that,  following  contact  with  a  large 
number  of  moderately  sensitized  corpuscles,  a  fluid  containing 
alexin  may  subsequently  act  on  similar  corpuscles  as  if  no  active 
substance  were  present,  but,  on  more  highly  sensitized  corpuscles  of 
the  same  species,  as  if  still  present.  It  will  scarcely  be  claimed  that 
the  alexin  used  up  by  a  feeble  sensitization  differs  from  the  one  taken 
by  a  greater  sensitization.  Such,  however,  is  apparently  the  con- 
clusion of  Remy.*  This  investigator  mixes  sensitized  typhoid 
bacilli  with  his  alexin  and  finds  that  subsequently  added  cor- 
puscles remain  intact  when  sensitized  by  a  serum  of  moderate 
activity,  whereas  they  are  destroyed  if  the  serum  with  which  they 
have  been  treated  is  very  powerful;  from  this  he  concludes  that 
the  bacteriolytic  alexin  differs  from  the  hemolytic  alexin.  He 
might  equally  well  have  concluded  that  there  is  an  alexin  partic- 
ularly adapted  for  heavily  sensitized  corpuscles,  since  such  cor- 
puscles are  hemolyzed  in  a  fluid  in  which  the  same  corpuscles, 
more  weakly  sensitized,  remain  intact.  In  fact,  Remy  has  usnl  a 
hemolytic  serum  that  has  a  higher  sensitizing  power  than  his  anti- 
typhoid serum. 

*  Contribution  &  l'e"tude  des  substances  actives  du  seYum.     Bull,  de  I'Academie 
de  Mddecine  de  Belgique,  1903. 


ALEXIN  ABSORPTION.  407 

Similar  instances  have  been  met  with  in  the  studies  on  whooping- 
cough  made  by  one  of  us  with  Gengou.  The  sera  of  three  children — 
a  brother  and  two  sisters  —  suffering  from  the  disease  were  tested 
on  the  same  day  and  in  the  same  amounts ;  as  a  general  thing  we 
used  highly  sensitized  corpuscles  to  test  for  alexin  fixation.  In  the 
control  containing  normal  human  serum,  alexin  and  bacteria, 
hemolysis  appeared  in  a  few  minutes ;  it  took  one-half  hour  and  one 
hour  respectively  in  tubes  containing  sera  of  two  of  the  children;  with 
the  third  serum  there  was  no  hemolysis.  The  three  whooping-cough 
sera,  then,  were  unequally  active,  and  it  was  found  that  the  most 
active  came  from  the  child  that  first  fell  ill  and  was  then  convales- 
cent, while  the  weakest  was  from  the  one  that  was  last  taken  with 
the  disease  and  still  showed  marked  symptoms.  The  result  is  quite 
natural,  but  if  the  sensitizing  power  were  only  moderate  even  in 
recovered  children  one  might  wrongfully  be  led  to  the  conclusion 
that  the  alexin  that  affects  the  whooping-cough  bacillus  differs  from 
the  hemolytic  alexin. 

As  a  matter  of  fact  the  method  is  very  exact  only  in  the  demon- 
stration of  powerful  sensitizers,*  and  is  not  applicable  for  the  ac- 
curate titration  of  the  activity  of  antimicrobial  sera,  or,  at  least, 
cannot  be  compared  in  exactitude  to  Ehrlich's  methods  employed 
in  measuring  the  potency  of  antitoxins. 

And  indeed  the  originators  of  the  method  recommended  it 
rather  for  the  qualitative  study  of  sera  than  for  a  quantitative 
evaluation  of  their  activity. 

*** 

The  researches  of  Pfeiffer  and  Friedbergerf  and  of  Sachs  $  con- 
cerning the  antagonistic  properties  (antibacteriolytic  or  antihe- 
molytic)  of  normal  sera  may  now  be  considered. 

A  few  data  in  hemolysis  will  simplify  the  matter  in  hand.     A 

*  It  is  not  surprising,  then,  that  in  Moreschi's  experiments  (Berlin,  klin.  Woch., 
1906,  p.  1244)  the  method  failed  to  demonstrate  sensitizing  properties  in  the 
serum  of  a  man  who  had  received  a  single  injection  of  killed  tyhoid  bacilli  a  few 
days  previously. 

t  Pfeiffer  and  Friedberger,  Deutsche  med.  Woch.,  1905,  Nos.  1  and  29;  Cen- 
tralblatt  f.  Bakt.,  XLI,  1906. 

t  Sachs,  Deutsche  medicin.  Wochen.,  1905,  No.  18;  Centralblatt  f.  Bakt., 
XL,  1906. 


408  STUDIES  IN   IMMUNITY. 

serum,  whether  normal  or  immune,  may  be  endowed  with  sensitizing 
power  for  certain  corpuscles  and  consequently  may,  in  their  presence, 
produce  alexin  fixation, although  as  serum  it  tends  simply  to  inhibit 
such  a  fixation.  These  two  forces  in  the  same  serum  oppose  one 
another  and  the  antagonistic  property  will  be  the  more  evident 
the  weaker  the  sensitizing  property.  Under  such  conditions  it 
might  well  happen  that  there  would  be  no  hemolysis,  in  other 
words,  it  might  appear  as  if  no  sensitizer  were  present.  So,  for 
example,  it  is  generally  considered  that  normal  rabbit  serum  (56 
degrees)  does  not  sensitize  bovine  corpuscles;  in  a  mixture  of 
bovine  corpuscles  (0.05  c.c.),  heated  rabbit  serum  (0.5  c.c.),  and  a 
little  guinea-pig  alexin  (0.1  c.c)  there  is  indeed  no  hemolysis.  But 
if  the  corpuscles  are  first  subjected  to  rabbit  serum,  then  centri- 
fugalized,  the  supernatant  fluid  decanted,  and  an  equal  amount 
of  normal  salt  solution  added,  followed  by  alexin,*  we  find  that 
slow  but  complete  hemolysis  takes  place.  Normal  rabbit  serum, 
then,  is  sensitizing  for  bovine  corpuscles,  although  weakly  so. 

Conversely,  when  corpuscles  that  are  strongly  sensitized  by  this 
serum  are  chosen,  as  Sachs  has  done,t  there  is  no  danger  of  losing 
sight  of  the  antagonistic  property.  It  is  found,  for  example,  that 
normal  rabbit  serum  sensitizes  goat  corpuscles  strongly.  We  mix 
0.6  c.c.  of  rabbit  serum  (56  degrees)  with  0.2  c.c.  of  salt  solution 
containing  25  per  cent  of  washed  goat  blood;  half  an  hour 
later  0.05  c.c.  of  fresh  guinea-pig  serum  is  added.  Hemolysis  is 
nearly  complete  in  one  and  a  half  hours  at  37  degrees,  whereas 
in  a  control  with  salt  solution  replacing  the  rabbit  serum  there 
is  no  hemolysis. 

In  this  case  the  antagonistic  property,  although  present,  is  con- 
cealed. Although  it  fails  to  prevent  hemolysis,  it  does  retard  it 
notably.  If  we  make  a  mixture  of  goat  blood  and  rabbit  serum  like 
the  preceding  and,  after  contact,  centrifugalize,  remove  the  serum, 
and  replace  it  with  salt  solution,  we  find  that  the  addition  of  alexin 
will  produce  a  much  more  rapid  hemolysis;  it  is  complete,  indeed, 
in  from  15  to  20  minutes.  By  removing  the  serum  we  have  eliminated 
the  antagonistic  property  and  hemolysis  is  accelerated.  As  is  evi- 

*  A  control  is  made  at  the  same  time  with  corpuscles  that  have  not  been  treated 
with  rabbit  serum  to  which  salt  solution  and  alexin  are  added. 

t  Analogous  results  have  been  obtained  in  bacteriolysis  by  Pfeiffer  and 
Fried  berger. 


ALEXIN  ABSORPTION.  409 

denced  by  this  experiment,  the  antagonistic  property  inhibits  the 
hemolysis  produced  by  a  normal  sensitizer.* 

Although  the  antagonistic  power  prevents  hemolysis  in  a  mixture 
of  bovine  corpuscles,  normal  rabbit  serum,  56  degrees,  and  guinea- 
pig  alexin,  because  the  serum  is  only  faintly  sensitizing  for  the 
corpuscles,  there  is  no  inhibition  when  goat  corpuscles  are  used,  be- 
cause they  are  powerfully  sensitized  by  the  rabbit  serum.  In  the 
latter  case,  as  we  have  just  seen,  the  antagonistic  property  simply 
retards  hemolysis.  This  effect  may  be  shown  in  another  way,  as 
is  evidenced  by  the  experiments  of  Pfeiffer  and  Friedberger  and  of 
Sachs.  In  their  experiments  the  property  that  opposes  the  antag- 
onistic property,  namely,  the  sensitizer,  is  removed.  After  treating 
normal  rabbit  serum  (56  degrees)  with  a  sufficient  amount  of  goat 
corpuscles  we  have  a  fluid  that  is  no  longer  sensitizing,  but  purely 
antagonistic;  it  will  therefore  protect  moderately  sensitized  goat 
corpuscles  from  hemolysis. 

If  the  sensitizer  (whether  in  an  immune  serum  or  normal  serum) 
is  too  strong,  the  antagonistic  property  will  be  overcome  and  hemoly- 
sis take  place.  The  origin  of  the  sensitizer  is  immaterial,  but  its 
strength  is  all-important. 

This  fact  is  quite  conceivable  in  view  of  the  fact  that  the  sensitizer 
does  not  unite  directly  with  the  alexin.  It  is  the  sensitized  cor- 
puscle that  unites  with  the  alexin,  and  the  greater  the  sensitization 
the  better  this  union.  The  union  with  the  sensitizer  changes  the 
properties  of  molecular  adhesion  in  the  corpuscle  so  that  its  avidity 
for  the  alexin  is  increased ;  in  a  similar  manner  the  action  of  agglu- 
tinins  on  bacteria  is  to  increase  their  reaction  to  the  clumping 
effect  of  electrolytes.  Any  result  produced  by  the  sensitizer  depends, 
not  on  its  proper  nature,  but  on  its  effect. 

*  This  antagonistic  effect  is  so  distinct  that  goat  corpuscles  that  have  been 
sensitized  by  a  small  dose  of  rabbit  serum  and  then  suspended  in  salt  solution  are 
hemolyzed  by  alexin  more  rapidly  than  the  corpuscles  sensitized  by  twice  as  much 
serum  when  kept  in  this  serum.  For  example:  in  each  of  four  tubes  is  placed 
0.05  c.c.  of  goat  blood;  to  A  and  B  is  added  0.4  c.c.  of  normal  rabbit  serum, 
56  degrees  and  to  C  and  D  0.2  c.c.  of  the  same  serum.  After  2  hours'  contact 
A  and  C  are  centrifugalized,  the  supernatant  fluids  removed  and  0.4  and  0.2  c.c. 
of  salt  solution  added  to  the  sediments  in  A  and  C  respectively.  To  each  of  the 
four  tubes  is  then  added  0.1  c.c.  of  guinea-pig  alexin.  Hemolysis  is  complete  in 
A  in  1 5  minutes,  in  C  in  a  half  hour,  in  B  in  50  minutes,  and  in  D  in  a  little  over 
an  hour, 


410  STUDIES  IN  IMMUNITY. 

This,  to  be  sure,  is  not  the  opinion  of  Sachs,  who  believes  that 
there  is  inhibition  to  hemolysis  when  a  specific  serum  is  used,  and 
not  when  a  normal  sensitizer  like  normal  rabbit  serum  is  employed. 
In  harmony  with  Ehrlich's  theory  Sachs  considers  that  normal 
sensitizers  possess  a  complementophilic  arm  that  is  very  avid  of 
alexin  (complement) ;  even  more  so  indeed,  than  is  the  corresponding 
arm  in  an  immune  sensitizer.  When  acted  on  by  a  normal  sensiti- 
zer the  corpuscles  easily  fix  alexin  even  in  presence  of  an  excess  of 
normal  serum.  But  since  a  normal  serum  contains  several  normal 
sensitizers  (Ehrlich  and  his  pupils  presuppose  the  existence  of  very 
numerous  antibodies  even  in  normal  serum),  these  bodies  take  hold 
of  the  alexin  present  and,  having  a  superior  affinity  for  it,  prevent 
its  fixation  on  corpuscles  even  when  they  are  sensitized  by  an 
immune  serum.  The  antagonistic  property  therefore  would  be 
due  to  the  presence  in  serum  of  certain  normal  sensitizers  that 
have  no  affinity  for  the  corpuscles  employed,  but  monopolize  the 
complement. 

This  theory  is  irreconcilable  with  the  fact  that  the  addition  of 
salt  solution  suffices  to  attenuate  the  antagonistic  effect  of  serum 
to  a  marked  degree.  The  first  premise  of  the  hypothesis  is  incorrect ; 
the  antagonistic  property  is  also  present  when  a  normal  sensitizer 
is  used,  as  we  have  just  seen,  and  as  the  following  experiment  further 
evidences : 

Two  series  of  mixtures  are  prepared  at  the  same  time.  The  first 
series  comprises  eight  tubes,  each  containing  1  c.c.  of  a  5  per  cent 
suspension  of  washed  goat  corpuscles.  To  four  of  these  tubes 
normal  sensitizer,  that  is  to  say,  normal  rabbit  serum,  56  degrees, 
is  added  in  varying  amounts  (0.4,  0.2,  0.1  and  0.05  of  a  cubic 
centimeter) ;  to  the  other  four  a  specific  sensitizer  (serum  of  a  rab- 
bit immunized  against  goat  blood)  in  doses  of  0.01,  0.005,  0.003  + 
and  0.0025  of  a  cubic  centimeter. 

One  hour  later  the  tubes  are  filled  with  salt  solution  centrifugal- 
ized,  the  supernatant  fluids  decanted,  and  0.6  of  a  cubic. centimeter 
of  salt  solution  added  to  each  sediment.  To  each  tube  is  then 
added  0.05  of  a  cubic  centimeter  of  fresh  guinea-pig  serum  (alexin). 

In  the  second  series  of  tubes  the  same  steps  are  taken  except 
that,  after  sensitization,  centrifugalization  and  decanting,  0.1  of  a 
cubic  centimeter  of  salt  solution  plus  0.5  of  a  cubic  centimeter  of 


ALEXIN  ABSORPTION. 


411 


heated  rabbit  serum  deprived  of  its  sensitizer  by  contact  with  goat 
corpusles  is  added.*  The  alexin  is  then  added  as  to  the  first  series. 

Controls  are  also  made  without  any  sensitizer,  either  normal  or 
immune,  to  prove  the  necessity  of  its  presence  for  the  production 
of  hemolysis. 

In  the  following  table  is  shown  the  length  of  time  in  minutes 
required  for  complete  hemolysis  at  35°  C.  The  letters  "M,"  "SI" 
and  "Nul"  indicate  that  after  several  hours  hemolysis  is  marked, 
slight  or  none,  as  the  case  may  be. 


OCCURRENCE    OF    HEMOLYSIS. 


Corpuscles  sus- 
pended in: 

Corpuscles  sensitized  by: 

Non-sen- 
sitized cor- 
puscles. 

NaClsol  ,0.6c.c 
+  alexin,  0.05 
c.c  

Immune  serum. 

Normal  serum. 

0.0025 

0.003 

0.005 

0.01 

0.05 

0.1 

0.2 

04 

Nul 

32' 
SI. 

20' 

SI. 

15' 
M. 

7' 
120' 

100' 
Nul 

25' 

SI. 

15' 
M. 

10' 
M. 

Nul 
Nul 

NaClsoL.O.lc.c 
4-  normal  rab- 
bit   serum 
minus     sensi- 
tizer, 0.5  c.c.+ 
alexin,  0.05c.c. 

In  the  mixtures  containing  salt  solution  but  no  antagonistic  serum 
we  may  estimate  the  relative  potency  of  the  two  sensitizers;  we 
find,  for  example,  that  0.005  of  a  cubic  centimeter  of  immune  serum 
affects  the  corpuscles  as  much  as  a  relatively  large  dose  (0.2  of  a 
cubic  centimeter)  of  normal  rabbit  serum,  and  that  0.01  of  a  cubic 
centimeter  of  immune  serum  sensitizes  a  little  better  than  does  0.4 
of  a  cubic  centimeter  of  normal  serum,  and  so  forth.  It  is  further 
to  be  noted  that  the  antagonistic  serum  (deprived  of  sensitizer) 
retards  hemolysis  approximately  equally  in  corpuscles  sensitized 
to  the  same  degree  by  normal  or  by  immune  serum. 

*  To  the  sediment  of  5  c.c.  of  washed  goat  blood  5  c.c.  of  rabbit  serum,  56 
degrees,  was  added.  A  few  hours  later  the  mixture  was  centrifugalized  and  the 
supernatant  fluid  removed. 


412  STUDIES  IN   IMMUNITY. 

Before  Sachs'  observations,  Pfeiffer  and  Friedberger  had  studied 
the  inhibiting  effect  of  normal  serum  on  bacteriolysis  of  the  cholera 
vibrio.  Their  sensitizer,  which  was  very  active  against  the  vibrio, 
was  first  removed  by  treating  the  immune  serum  with  vibrios, 
followed  by  centrifugalization  and  decanting  of  the  serum:  the 
supernatant  fluid  so  obtained,  on  injection  into  the  peritoneal  cavity 
of  a  normal  animal  together  with  vibrios  sensitized  by  a  moderate 
dose  of  anticholera  serum,  prevents  bacteriolysis.  Pfeiffer  and 
Friedberger  further  noted  that  for  the  antagonistic  power  to  be 
manifest  a  moderate  sensitization  only  should  be  used.  They 
have  studied  the  conditions  affecting  the  phenomenon  with  great 
care  and  reserve  any  decision  as  to  its  interpretation. 

Sachs'  findings  in  hemolysis  are  analogous  to  those  of  Pfeiffer 
and  Friedberger.  There  can  be  no  doubt  that  any  explanation 
for  one  will  serve  for  the  other;  the  inhibiting  property  of  certain 
constituents  of  serum  on  the  fixation  of  alexin  on  sensitized  blood 
cells,  as  opposed  to  salt  solution,  is  doubtless  applicable  to  bacte- 
riolysis as  well.  A  single  objection  occurs :  when  faintly  sensitized 
vibrios  are  injected  into  the  peritoneal  cavity  bacteriolysis  does  or 
does  not  take  place,  depending  on  whether  antagonistic  serum  has 
or  has  not  been  previously  added.  But  why  is  such  serum  necessary 
to  protect  the  vibrios?  Does  not  the  peritoneal  cavity  contain  a 
certain  amount  of  an  exudate  which  resembles  the  antagonistic 
serum  at  least  more  closely  than  it  does  salt  solution?  Why,  then, 
does  not  this  exudate  inhibit  bacteriolysis? 

We  find  by  experiment  that  peritoneal  exudate,  even  when  heated, 
has  no  more  inhibiting  effect  on  hemolysis*  than  salt  solution.  For 
example,  a  little  peritoneal  exudate  from  a  rabbit  was  heated  to 
56  degrees.  At  the  same  time  the  serum  of  the  same  animal  was 
obtained  and  treated  in  the  same  manner.  One-twentieth  of  a 
cubic  centimeter  of  moderately  sensitized  bovine  blood  (rabbit  >  ox 
serum)  is  placed  in  each  of  several  tubes,  and  0.3  of  a  cubic  centi- 
meter of  salt  solution,  normal  serum  or  peritoneal  exudate,  respec- 
tively, is  added;  0.05  of  a  cubic  centimeter  of  fresh  guinea-pig 
serum  is  then  added  to  each  tube.  Hemolysis  appears  rapidly  in 
the  mixture  containing  salt  solution  and  almost  as  soon  in  the  one 

*  It  seems  that  from  the  close  analogies  between  bacteriolysis  and  hemolysis 
any  conclusions  referring  to  one  are  applicable  to  the  other. 


ALEXIN  ABSORPTION.  413 

with  exudate,  whereas  it  is  very  much  retarded  by  the  normal 
serum. 

Peritoneal  exudate,  then,  favors  the  demonstration  of  alexic 
activity.  Thirteen  years  ago  the  "  reactivation"  of  heated  cholera 
serum  by  fresh  normal  serum  was  demonstrated  by  one  of  us.  It 
was  also  shown  at  the  time  that  bacteriolysis  is  due  to  the  collabo- 
ration of  the  specific  antibody  and  the  alexin.  The  granular  trans- 
formation of  vibrios,  which  is  indicative  of  bactericidal  power, 
generally  occurs  more  rapidly  in  the  peritoneal  cavity  than  in 
mixtures  in  vitro  containing  serum.  The  reason  for  this  is  simply 
that  the  antagonistic  property  of  heated  serum  retards  bacteriolysis 
more  markedly  in  vitro  than  in  vivo. 

It  is  evident  that  the  presence  of  an  antagonistic  property  must 
have  been  a  great  source  of  error  in  many  experiments  and  in 
particular  in  those  based  on  the  principle  of  specific  absorption 
and  those  dealing  with  the  multiplicity  of  active  substances  in  a 
given  serum;  obvious  examples  of  this  error  are  present  in  experi- 
ments dealing  with  the  discussion  concerning  the  unity  or  multi- 
plicity of  alexins. 


XXII.  A  CONTRIBUTION  TO  THE  STUDY  OF  MOLECULAR 
ADHESION  WITH  A  CONSIDERATION  OF  ITS  FUNC- 
TION IN  VARIOUS  BIOLOGICAL  PHENOMENA  * 

BY  DR.   GENGOU. 

L 

For  some  years  biologists  and  bacteriologists  have  studied  both 
the  inorganic  and  the  organic  colloidal  substances.  As  we  know 
the  majority  of  the  writers  consider  the  sols  (hydrosols,  etc.),  that 
is  to  say,  the  "solutions"  of  colloidal  substances  in  fluid,  as  being 
not  true  solutions,  but  ultramicroscopic  or  microscopic  suspensions 
of  colloidal  particles  in  the  fluid.  In  addition  to  colloidal  solutions 
we  have  fine  suspensions,  such  as  mastic,  aqueous  gum  arabic,  etc., 
which  consist  of  fine  particles  in  a  fluid,  with  the  difference  that 
these  particles  are  visible. 

We  have  learned  a  great  deal  about  colloidal  solutions  from 
studying  these  fine  suspensions,  inasmuch  as  both  substances  have 
certain  properties  in  common.  We  believe  also  that  certain  studies 
of  the  chemical  precipitates  which  sediment  easily  are  bound  to 
facilitate  our  knowledge  of  the  various  manifestations  of  colloidal 
substances.  Inasmuch  as  colloids  are  probably  very  fine  suspen- 
sions, it  is  likely  that  all  transitions  between  them  and  the  sedi- 
menting  chemical  precipitates  with  large  particles,  exist.  We 
must  admit  that  all  these  substances  in  suspension  have  certain 
common  properties.  It  is  often  easier  to  work  with  chemical 
precipitates  than  with  colloidal  solutions,  and  such  a  method  is  there- 
fore advantageous  in  studying  those  properties  which  are  common 
to  both  substances. 

Among  these  common  properties  the  one  which  we  shall  consider 
almost  entirely  is  the  power  of  adsorption.  We  have  long  known 
that  certain  solid  substances  in  the  form  of  a  fine  powder  absorb 

*  This  article  is  a  re"sume\  which  was  kindly  furnished  by  Dr.  Gengou,  of  an 
article  on  the  subject  which  appeared  in  the  Archives  Internat.  de  Physiologic, 

Vol.  7,  Fasc.  1  and  2,  1908. 

414 


STUDY  OF  MOLECULAR  ADHESION.  415 

substances  in  the  form  of  gases  or  solutions  (electrolytes,  colloidal 
sols).  These  adsorption  phenomena  by  solid  bodies  bear  a  definite 
relation  to  the  considerable  surface  development  of  the  particles 
of  these  substances.  Colloids  in  the  state  of  gels,  which  also  have  a 
large  surface,  are  likewise  endowed  with  energetic  adsorption 
properties.*  The  laws  of  adsorption  by  solid  bodies  are  the  same 
as  with  colloidal  gels. 

The  phenomena  of  molecular  adhesion  may  begin  between  two 
substances  of  different  natures  and  may  also  occur  between  the 
particles  of  the  same  substance,  for  example,  the  particles  of  an 
insoluble  solid  suspended  in  water.  If  increasing  amounts  of  a 
suspension  of  barium  sulphate  in  water  are  added  to  a  constant 
volume  of  water,  we  find  that  the  mixture  becomes  cleared  of  the 
powder  with  a  rapidity  which  varies  directly  with  the  amount  of 
barium  sulphate  added.  Naturally  this  is  due  to  the  fact  that  the 
nearer  the  particles  are  to  one  another  the  more  their  mutual  ad- 
hesion is  facilitated  and  they  form  clumps  which  sediment  more 
easily.  The  sedimentation  of  a  substance  which  is  insoluble  in 
water  is,  as  we  know,  facilitated  by  certain  colloidal  solutions 
and  viscous  fluids.!  A  few  years  ago  Mullerf  studied  the  inhibit- 

*  Van  Bemmelen,  Zeitschrift  fur  Anorg.  Chem.,  1900,  23.  This  author  has 
suggested  the  name  of  adsorption  to  designate  the  accumulation  of  gas  or  fluids 
in  porous  bodies  or  on  the  surface  of  non-porous  bodies,  and  the  word  absorption 
for  the  instances  in  which  the  molecules  of  the  adsorbed  substance  interchange 
with  those  of  the  adsorbing  substance,  for  example,  homogeneous  solutions  of 
gases,  liquids  or  solids.  The  phenomena  which  we  are  about  to  consider  deal  with 
the  adhesion  of  various  substances  to  solid  bodies  suspended  in  a  fluid,  and  we 
prefer  to  use  the  term  adsorption. 

t  Lobny  de  Bruyn  (Rec.  des  Trav.  chim.  des  Pays  Bas,  1900,  vol.  19)  has 
shown  in  particular  that  if  solutions  of  salts  which  normally  form  an  insoluble 
precipitate  are  added  to  gelatin  the  sedimentation  occurs  much  more  slowly. 
The  reaction  between  these  salts,  however,  takes  place  just  as  well  under  these 
conditions,  for  the  rapidity  of  the  reaction  is  as  rapid  in  gels  as  in  water. 

(E.  Cohen,  Eder's  Jahresbericht  f.  Photographic,  1895,  cited  by  Hamburger, 
Osmot.  Druck  u.  lonenlehre,  vol.  3,  p.  89. 

Rothland,  Zeitschrift  f.  anorg.  Chem.,  vol.  40;  Spring,  Bull,  de  1'Acad.  Roy. 
de  Belg.,  1900,  p.  515.) 

Since  this  time  several  authors  have  applied  the  same  method  and  similar 
.  methods  in  preparing  colloidal  solutions  of  substances  that  are  usually  insoluble. 

Paal  and  Amberger:  Bericht  d.  deutschen  chem.  Gesellschaft,  vol.  XXXV  and 
XXXVII;  Paal  and  Voos,  Ibid,  vol.  XXXVII;  Lottermoser,  Ueber  anorg. 
Kolloide,  Stuttgart,  1901;  Guthier:  Zeitschrift  f.  anorg.  Chem.,  XXXII.  Hein- 
rich,  Bericht  d.  deutsche  chem.  Gesellschaft,  XXXVI;  Garbowski:  Ibid;  Carey 
Lea,  Ibid,  XXIV.) 

J  Bericht  d.  deutschen  chem.  Gesellschaft,  1904. 


416  STUDIES  IN  IMMUNITY. 

ing  action  of  various  stable  colloids  on  the  sedimentation  of  a 
colloidal  solution  of  gold  to  which  electrolytes  had  been  added  and 
thought  that  he  found  some  relation  between  the  viscosity  and  the 
intensity  of  its  protecting  power.  Victor  Henri  and  A.  Meyer* 
did  not  think  that  the  viscosity  was  a  sufficient  factor  to  explain  the 
suspension  of  chemical  precipitates  by  stable  colloids. 

We  have  studied  the  action  of  barium  sulphate  that  has  been 
washed  and  suspended  in  distilled  water  on  the  addition  of  various 
stable  colloidal  solutions  or  viscous  fluids.  Certain  colloidal  solu- 
tions agglutinate  barium  sulphate  energetically,  for  example,  a 
solution  of  starch  or  farina;  certain  organic  fluids  even  when  deprived 
of  their  cellular  elements  by  centrifugalization  (peritoneal  or  syno- 
vial  fluid,  nasal:  mucus,  saliva)  do  the  same  thing;  the  agglutin- 
ating substance  in  these  fluids  would  appear  to  be  mucin. 

The  majority  of  stable  colloids  and  viscous  fluids,  on  the  contrary, 
inhibit  the  sedimentation  of  barium  sulphate.  On  shaking  barium 
sulphate  in  aqueous  solutions  of  sugar,  glycerine,  gum  arabic,  gum 
tragacanth,  agar,  gelatin,  glue,  dextrin,  serin,  pseudoglobulin, 
euglobulin  or  fibrinoglobulin,  we  find  that  the  masses  of  barium 
sulphate  break  up  into  much  smaller  particles  of  apparently  uni- 
form size  which  have  slight  tendency  to  adhere  to  one  another. 
As  a  result,  we  obtain  much  finer  suspensions,  which  are  more 
homogeneous  and  stable  and,  in  the  case  of  barium  sulphate,  are  of 
milky  appearance.  These  suspensions,  when  left  to  themselves, 
sediment  very  slowly. 

It  is  only  rarely  that  the  action  of  such  solutions  on  barium  sul- 
phate is  due  to  their  viscosity.  Syrups  composed  of  sugar  or 
glycerine  do  act  by  viscosity;  the  more  marked  this  viscosity  is,  the 
slower  the  sedimentation  of  the  barium  sulphate.  All  the  other  sub- 
stances which  we  have  just  mentioned,  however,  as  dissociating 
barium  sulphate,  do  not  act  through  their  viscosity  unless  much 
more  concentrated  solutions  than  necessary  are  employed.  Vis- 
cosity, then,  functions  only  in  a  subsidiary  manner.  Thus  we  find 
that  a  gum  solution  of  0.03  per  cent  in  distilled  water  which  reacts 
with  Ostwald's  viscosimeter  just  as  distilled  water  does,  suffices, 
notwithstanding,  to  hold  barium  sulphate  in  suspension. 

The  mechanism  of  the  suspension  of  barium  sulphate  becomes 
*  Revue  gen.  des  Sciences,  1904. 


STUDY  OF  MOLECULAR  ADHESION.  417 

evident  when  the  suspension  which  has  been  in  contact  with  these 
various  colloidal  solutions  is  repeatedly  washed.  If  this  washing 
is  kept  up  until  no  detectable  amount  of  the  colloid  remains,  we  find 
that  the  treated  suspension,  when  placed  in  distilled  water,  remains 
suspended  as  well  as  if  the  colloidal  solution  were  still  present. 
This  fact  is  brought  out  very  clearly  on  employing  gum  arabic, 
agar,  gelatin,  gum  tragacanth,  glue  or  pseudoglobulin.  It  seems 
reasonable  to  believe  then,  that  barium  sulphate,  when  treated  with 
solutions  of  these  substances,  absorbs  something  from  them  and 
forms  with  the  colloid  a  complex  which  is  sufficiently  stable  to 
resist  numerous  washings  with  distilled  water.*  Such  is  not  the 
case  with  the  suspension  that  has  been  treated  with  sugar  or  glycer- 
ine syrup.  A  suspension  treated  in  this  way  after  washing  becomes 
like  untreated  suspension;  it  takes  nothing  from  these  solutions 
which  hold  it  in  suspension  through  their  viscosity. 

Serum  albumin,  euglobulin  and  fibrinoglobulin  also  form  com- 
plexes with  barium  sulphate;  these  complexes,  however,  withstand 
washing  poorly. 

*** 

It  is  easily  demonstrable  that  a  complex  between  the  insoluble 
barium  sulphate  and  the  colloids  is  formed :  a  weak  solution  of  gum 
arabic  treated  with  a  sufficient  quantity  of  barium  sulphate  and 
then  freed  of  it  by  centrifugalization  is  no  longer  able  to  dissem- 
inate fresh  barium  sulphate. 

If,  on  the  other  hand,  a  substance  such  as  calcium  phosphate, 
which  is  easily  dissolved  in  acetic  acid,  is  used  instead  of  barium 
sulphate,  the  adsorbed  colloid  may  be  liberated  from  the  complex 
after  washing  by  dissolving  the  adsorbing  substance;  the  colloid 
liberated  in  this  manner  is  demonstrable  by  the  fact  that  it  is  able 
to  disseminate  fresh  barium  sulphate. 

When  a  washed  complex,  such  as  barium  sulphate  plus  agar,  is 

*  Substances  other  than  barium  sulphate,  for  example,  animal  charcoal,  lead 
iodide,  calcium  oxalate,  mercurous  sulphate,  manganese  carbonate  and  copper 
oxide  are  held  in  suspension  more  or  less  well  by  gum  arabic.  Gum  arabic, 
however,  does  not  succeed  in  dissociating  fine  particles  of  kaolin,  chromate  of 
silver,  sulphate  of  calcium  and  sulphate  of  zinc.  It  seems  reasonable  that  such 
should  be  the  case,  since  we  are  dealing  with  various  powdered  substances,  the 
adsorbing  power  of  which  will  evidently  not  be  the  same  for  the  colloid  under 
consideration. 


418  STUDIES  IN  IMMUNITY. 

left  for  a  long  time  at  room  temperature,  that  is,  for  several  days,  a 
small  amount  of  the  colloidal  substance  is  demonstrable,  after 
centrifugalization,  in  the  supernatant  fluid.  Certain  factors  such 
as  heat  and  the  like  accelerate  the  breaking  up  of  these  complexes. 
Thus  we  find  that  a  complex  of  barium  sulphate  and  gum  arabic 
which  has  been  washed,  and  has  remained  for  several  days  at  room 
temperature  and  has  liberated  very  little  of  its  colloid,  liberates 
much  more  of  it  when  boiled  for  a  few  moments.* 

The  adsorption  of  a  colloid  by  an  insoluble  substance  has  long 
been  known.  The  relation  between  this  adsorption  and  the  dis- 
semination of  insoluble  substances  by  colloidal  media  is  what 
gives  interest  to  our  data. 

Although  it  may  seem  quite  evident  from  what  we  have  said  that 
the  facts  observed  are  identical  with  adsorption  phenomena,  we 
have,  nevertheless,  endeavored  to  establish  this  identity  more  com- 
pletely. Physicists  have  shown  that  if  they  add  identical  volumes 
of  a  fluid  containing  adsorbable  substances  in  increasing  concentra- 
tion to  a  fixed  quantity  of  the  adsorbing  substance,  that  the  absolute 
amount  of  the  substance  adsorbed  increases  up  to  a  certain  point 
with  the  concentration,  but  not  in  direct  proportion  to  it.  It  does 
not  always  form  the  same  fraction  of  the  quantity  of  adsorbable 
substance  present.  We  find  experimentally  that  the  denominator 
of  this  fraction  increases  in  proportion  to  the  increase  in  concentra- 
tion, so  that  the  proportion  of  substance  adsorbed  relative  to  the 
amount  of  adsorbable  substance  employed,  diminishes. 

The  same  rule  is  true  in  the  adsorption  of  gum  arabic  by  barium 
sulphate;  on  the  addition  of  increasing  doses  of  gum  arabic  in  a 
given  volume  to  the  same  amount  of  this  suspension,  larger  and 
larger  amounts  of  the  colloid  are  adsorbed ;  the  fraction  of  the  colloid 
adsorbed,  however,  diminishes  in  proportion  to  the  total  amount 
of  colloid  present. 

In  this  relation  we  may  recall  the  careful  researches  of  Eisenberg 
and  Volk,t  who  observed  similar  effects  in  determining  the  amounts 
of  agglutinin  that  a  given  amount  of  bacteria  adsorbs  when  added 
to  increasing  doses  of  agglutinin  in  a  constant  volume.  This  fact 

*  Van  Bemmelen  has  shown  also  that  adsorption  takes  place  better  in  the 
cold  than  at  higher  temperatures;  in  our  instances  we  are  dealing  with  an 
adsorption  that  has  taken  place  in  the  cold  and  is  partially  broken  up  by  heat. 

t  Zeitschrift  fur  Hygiene,  vol.  40. 


STUDY  OF  MOLECULAR  ADHESION.  419 

is  one  of  those  most  strongly  indicative  of  the  relation  which  would 
seem  to  exist  between  adsorption  phenomena  and  the  reactions 
between  agglutinins  and  bacteria. 

The  suspension  of  an  inorganic  powder  in  a  colloidal  medium 
begins,  then,  by  adhesion  of  the  colloid  to  the  powder.  We  have 
just  seen  that  certain  colloids  on  uniting  with  barium  sulphate 
agglutinate  it.  The  two  phenomena  of  agglutination  and  dissocia- 
tion of  barium  sulphate  by  stable  colloids,  although  apparently  so 
different,  are  in  fact  due  to  the  same  mechanism,  namely,  the  adhe- 
sion of  the  colloid  to  the  suspension. 

We  have  already  found  that  a  given  colloidal  substance  in  uniting 
with  a  given  suspension  may  produce  either  its  dissociation  or  its 
agglutination.  This  is  particularly  the  case  with  such  colloids  as 
gelatin,  the  concentrated  solutions  of  which  harden  in  the  cold. 
The  dissemination  or  the  agglutination  of  a  suspension  of  barium 
sulphate  by  a  0.5  per  cent  solution  of  agar  depends  on  whether 
the  mixture  is  made  at  50°  or  at  16°  C.  In  both  instances 
adhesion  takes  place.  It  is  probable  that  weak  solutions  of  agar 
at  high  temperature  are  composed  of  very  small  particles  of  the 
substance,  which  particles  clump  together  as  the  temperature  is 
lowered.  This  supposition  indeed  finds  an  experimental  basis 
in  the  facts  that  Hardy*  has  noted.  This  author  found  that  solu- 
tions of  agar  and  gelatin  are  composed  of  granules,  the  volume  and 
disposition  of  which  depend  on  the  temperature.  The  effect  of 
agar,  then,  on  barium  sulphate  depends  on  its  physical  condition 
or  on  the  size  of  its  particles,  which  latter  fact  depends  on  the 
temperature  and  the  duration  of  time  that  the  colloid  has  remained 
at  this  temperature. 

The  effect  of  cold  on  agar  corresponds  to  the  effect  of  heat  on  the 
protein  substances  of  serum;  these  substances  when  unheated  are 
in  a  colloidal  condition  which  permits  them  to  adhere  to  barium 
sulphate  and  to  disseminate  it;  when  larger  proteid  particles  have 
been  produced  by  heating  the  clumping  of  the  sulphate  follows.! 

These  facts,  we  believe,  may  be  compared  with  another  phenom- 
enon :  if  a  fresh  amount  of  barium-sulphate  suspension  is  added  to 
a  complex  of  washed  barium  sulphate  and  colloid  (for  example, 
barium  sulphate  plus  gum  arabic),  complete  agglutination  of  the 

*  Proceedings  of  the  Royal  Society,  1900,  LXVI.  f  Gengou,  p.  326. 


420  STUDIES   IN  IMMUNITY. 

complex  plus  the  fresh  suspension  takes  place.  The  particles  of 
the  complex  probably  act  on  fresh  barium  sulphate  just  as  volu- 
minous particles  of  the  gum  would  act  on  fresh  barium  sulphate,  or, 
in  other  words,  as  do  the  granules  of  cold  solutions  of  gelatin  or  the 
albuminous  clumps  produced  in  serum  by  heating. 

*** 

With  the  exception  of  the  instances  in  which  the  viscosity  of  the 
fluid  intervenes,  the  dissemination  of  suspensions  in  the  solutions 
we  have  studied  is  due  to  a  substitution  of  the  adhesion  of  the 
particles  among  themselves  for  an  adhesion  of  the  particles  of  the 
suspension  to  particles  of  the  disseminating  colloid. 

The  dissemination  of  a  washed  suspension  by  colloidal  solutions 
resembles  the  inhibition  of  the  appearance  or  sedimentation  of 
precipitates  due  to  chemical  reactions  which  might  occur  in  such 
solutions.  We  think  that  the  facts  observed  by  Lobny  de  Bruyn 
and  others  in  this  connection,  which  have  been  made  use  of  to  obtain 
colloidal  solutions  of  substances  which  are  normally  insoluble,  are 
due  to  the  fact  that  as  soon  as  the  chemical  reactions  which  lead 
to  the  production  of  these  substances  within  a  stable  colloid  solu- 
tion are  finished,  there  is  established,  between  the  particles  of 
these  substances  and  the  neighboring  particles  of  the  surrounding 
colloid,  a  complex  which  is  similar  to  those  that  we  have  studied. 

Colloidal  solutions  obtained  in  this  way  are  therefore  suspen- 
sions in  a  greater  or  less  excess  of  stable  colloid  of  complexes 
formed  by  the  stable  colloid  with  an  insoluble  chemical  substance. 

The  dissemination  of  an  insoluble  salt  by  stable  colloids  in  the 
form  of  a  complex  should  apparently  be  compared  with  the  homo- 
geneous condition  which  colloids  give  to  emulsions  of  oil.  This 
phenomenon,  as  Quincke*  has  shown,  is  due  to  the  fact  that  these 
colloids  diminish  the  surface  tension  of  the  oil  droplets  for  water 
by  intervening  between  the  droplets  of  oil  and  those  of  water.  It  is 
probable  that  the  dissemination  of  barium  sulphate  in  a  colloidal 
medium  is  likewise  due  to  the  fact  that  the  surface  tension  which 
exists  between  the  suspension  and  water  is  lowered  by  the  presence 
of  the  colloid  on  the  surface  of  the  particles  of  suspension  that  have 
absorbed  it. 

*  Quincke,  Wiedemann's  Annalen,  1904,  XXXVII. 


STUDY   OF   MOLECULAR  ADHESION.  421 

Some  writers  have  concluded  from  Quincke's  experiments  that 
any  substance  which  diminishes  the  surface  tension  of  substance 
A  for  fluid  B  must  be  absorbed  by  A,  and,  on  the  other  hand,  that 
any  substance  that  has  been  adsorbed  must  diminish  the  surface 
tension  of  the  adsorbing  substance.  It  is  to  be  remembered, 
however,  that  in  certain  instances  the  mutual  adhesion  of  two 
substances  in  fine  suspension  in  a  fluid  results,  not  in  a  dis- 
semination, but  in  an  increase  of  surface  tension.  Thus  the 
mutual  flocculation  of  two  unstable  colloids  of  different  electric 
charges  indicates  an  increase  of  superficial  tension  resulting  from 
the  adhesion  of  particles  of  the  two  colloids,  which  fact  Biltz* 
correctly  compares  with  adsorption  phenomena. 

*** 

We  have  endeavored  to  determine  the  reaction  of  our  complexes 
of  barium  sulphate  plus  colloid  to  electrolytes.  Such  complexes 
as  we  have  already  mentioned  may  be  removed  from  the  colloidal 
solution  in  which  they  have  formed,  and  resuspended  in  distilled 
water.  We  have  stated  that  complexes  washed  in  this  manner 
react  to  electrolytes  and  flock  out  as  unstable  colloids  do  when 
these  electrolytes  are  present.  Alkalis,  and  particularly  acids, 
have  the  same  effect.  It  is  evident  that  in  this  case  we  are  dealing 
with  an  electrolytic  effect  and  not  with  a  phenomenon  of  plas- 
molysis  due  to  concentration  of  the  salt,  inasmuch  as  such  washed 
complexes  do  not  flocculate  when  the  salt  is  replaced  by  isotonic 
sugar  solution;  such  a  sugar  solution  may  be  employed  in  any 
concentration  without  flocculating  the  complex. 

This  flocculation  is  reversible,  inasmuch  as  when  the  salt  solution 
which  flocculates  is  removed  and  replaced  by  distilled  water,  the 
complex  recovers  its  original  appearance  and  is  again  dissemi- 
nated. Although  a  reversibility  occurs  in  the  agglutination  of 
certain  colloids  and  certain  fine  suspensions  by  means  of  salts  of 
alkalis  and  of  alkali  earth,  the  fact  should  be  mentioned  in  the 
present  instance.  We  might  suppose,  for  example,  that  in  pre- 
cipitating a  complex  of  barium  sulphate  plus  gum  or  the  like,  the 
electrolyte  simply  detaches  the  fixed  colloids  from  the  suspension, 
in  other  words,  breaks  up  the  complex  and  restores  barium  sul- 

*  Berichte  der  deutsche  chem.  Gesellschaft,  1904,  XXXVII. 


422  STUDIES  IN   IMMUNITY. 

phate  to  its  original  condition.  The  fact  that  the  flocculation  of 
the  complex  by  sodium  chloride  is  reversible  proves,  however, 
that  this  is  not  the  explanation ;  it  is  the  entire  complex  which  is 
flocculated. 

Flocculation,  on  the  contrary,  is  lacking  when  the  complex, 
barium  sulphate  plus  colloid,  has  not  been  washed  or  when,  after 
it  has  been  washed,  an  excess  of  stable  colloid  is  added.  This  latter 
colloid,  then,  would  seem  to  "protect"  complexes  just  as  it  protects 
an  unstable  colloid  from  flocculation  by  salts.  This  fact,  we  think, 
tends  to  validate  the  explanation  of  Beckhold,  Girard-Mangan  and 
v.  Henri,  Pauli,  and  others.*  They  propose  to  explain  the  mechan- 
ism of  the  protection  of  unstable  colloids  by  stable  colloids  against 
precipitation  by  electrolytes.  When  we  remove  a  complex  of 
barium  sulphate  plus  gum  from  the  colloidal  medium  in  which  it 
has  formed,  by  washing,  it  is  very  probable  that  we  do  not  obtain 
it  as  it  occurs  in  such  a  medium.  If  we  accept  the  conception  that 
has  been  proposed  by  Van  Bemmelen  for  the  phenomena  of  adsorp- 
tion, and  admit  that  the  preparation  of  emulsions  of  oil  in  gum 
solution  described  by  Quincke  (that  is,  the  covering  of  the  oil 
droplets  by  a  layer  of  gum),  is  similar  to  the  mode  of  prepara- 
tion of  our  complex,  we  may  suppose  that  the  particles  of  the  com- 
plex, barium  sulphate  plus  gum,  are  composed  of  a  center  formed 
by  the  barium  sulphate  around  which  the  particles  of  gum  are 
disposed  in  a  homogeneous  manner.  The  most  distant  of  these 
gum  particles  are  attracted  only  feebly  by  the  barium  sulphate 
and  become  detached  in  washing,  which  leaves  us  a  complex 
poorer  in  gum  than  in  its  original  condition.  We  may  then 
logically  imagine  that  on  placing  our  washed  complex  in  a  fresh 
gum  solution  it  becomes  covered  with  fresh  exterior  layers  of  gum 
which  have  been  removed  by  washing.  The  inhibiting  effect  of 
gum  solution  on  the  flocculation  of  the  complex  by  sodium 
chloride  may  then  be  due  to  an  increased  thickness  of  gum  and  to 
the  intervention  of  additional  superficial  layers  on  the  complex. 
The  following  fact  may  be  mentioned  in  support  of  this  concep- 
tion: if  complexes  of  barium  sulphate  and  gum  are  formed  in 
increasing  quantities  of  gum  solution  and  subsequently  washed 
in  distilled  water,  we  should  obtain  complexes  of  varying  richness 
*  Cited  by  Aron,  Bioch.  Centralblatt,  vol.  3. 


STUDY  OF   MOLECULAR  ADHESION.  423 

in  colloidal  substance,  the  flocculation  of  which  by  salts  should 
diminish  in  accordance  with  the  amount  of  colloid  present.  This 
fact  would  seem  to  us  to  demonstrate  that  it  is  the  stable  colloid 
adhering  to  the  powder  which  opposes  in  great  part  the  flocking 
action  of  the  electrolytes.  The  same  fact  also  probably  holds 
in  the  protection  by  a  stable  colloid  against  precipitation  of  an 
unstable  colloid  by  salts. 

*** 

Apart  from  the  flocculation  of  complexes  by  electrolytes,  all  the 
facts  described  to  the  present  are  due  to  the  same  cause,  namely, 
a  substitution  of  one  form  of  adhesion  for  another.  When  a  stable 
colloid  transforms  the  unstable  mixture  of  barium  sulphate  and 
water  into  a  much  more  delicate  and  stable  suspension,  it  is  due  to 
the  fact  that  one  adhesion  (barium  sulphate  plus  colloid)  is  sub- 
stituted for  another  adhesion  (barium  sulphate  plus  barium 
sulphate).  In  order  for  this  to  occur  the  attraction  of  the  suspen- 
sion for  the  colloid  must  naturally  be  stronger  than  the  attraction 
of  the  suspension  for  itself. 

Likewise  the  fact  that  a  stable  colloid  such  as  gum  or  serum 
disseminates  barium  sulphate  by  separating  the  mutual  adhesion 
of  the  particles  of  barium  sulphate  resembles  the  fact  that  a 
stable  colloid  may  inhibit  the  adhesion  of  barium  sulphate  to 
another  colloid.  Thus,  in  the  presence  of  serum,  starch  solution 
and  peritoneal  fluid  fail  to  agglutinate  barium  sulphate;  the  sus- 
pension adheres  to  the  colloids  of  the  serum  and  remains  dis- 
seminated in  spite  of  the  presence  of  the  agglutinating  colloids. 
In  the  same  way  a  stable  colloid  (gum)  prevents  the  mutual 
flocculation  of  fresh  barium  sulphate  with  a  washed  complex  of 
barium  sulphate  plus  gum.  In  this  instance  the  fresh  suspension 
adheres  to  the  free  stable  colloid  and  not  to  the  washed  complex. 

II. 

We  have  stated  that  barium  sulphate  may  be  held  in  fine  suspen- 
sion by  substances  other  than  stable  colloids.  Citrate  of  sodium, 
the  inhibiting  action  of  which  on  coagulation  of  the  blood  and  milk 
is  well  known,  will  do  this.  The  statements  we  have  made  about 
suspensions  of  powders  by  stable  colloids  render  unnecessary  any 


424  STUDIES  IN  IMMUNITY. 

full  description  of  the  action  of  citrate.  The  dissemination  of  barium 
sulphate  by  the  citrate  does  not  increase,  with  a  given  dose 
of  powder,  with  the  concentration  of  the  citrate.  We  have  found, 
for  example,  that  an  amount  of  powder  contained  in  eight  drops 
of  our  barium  sulphate  solution,  when  added  to  2  c.c.  of  fluid, 
is  well  disseminated  if  this  fluid  contains  from  0.005  per  cent 
to  0.1  per  cent  of  citrate.  Above  this  concentration  the  dissemi- 
nating property  of  the  solution  diminishes,  so  that,  in  20  or  30  per 
cent  citrate,  barium  sulphate  sediments  almost  as  rapidly  as  in 
distilled  water.  This  disseminating  action  may  also  occur  with 
other  substances  that  are  insoluble  in  water,  such  as  clay,  tri- 
calcium  phosphate,  animal  charcoal,  and  olive  oil. 

We  have  found  that  this  property  in  sodium  citrate  is  due  to  its 
acid  radicale.  If  increasing  doses  of  citric  acid  (0.0016  per  cent  to 
0.05  per  cent  in  our  experiments)  are  added  to  a  given  quantity  of 
barium  sulphate,  distinct  dissemination  takes  place  in  certain 
tubes,  as  indicated  by  a  suspension  of  the  powder.  As  with  sodium 
citrate,  the  disseminating  property  is  minimal  with  certain  doses 
of  citric  acid  and  diminishes  with  stronger  doses  of  it.  These 
facts  may  be  compared  with  the  recent  observations  of  Freund- 
lich  (Zeitschrift  fur  phys.  Chem.,  LVII,  4).  This  author  found  that 
charcoal  (Bliitkohle)  remains  for  a  long  time  in  suspension  in  methyl- 
amine,  dipropylamine,  trimethylamine,  pyridin,  codein  and  picric 
acid;  he  found  that  this  charcoal  suspension  has  an  optimum  in  the 
moderate  concentrations  of  these  substances.  Oechsner  de 
Coninck  and  Azalier  *  found  that  barium  sulphate  is  retained  in 
suspension  by  the  chlorhydrate  of  methylamine,  and  that  even 
when  it  is  warm  no  chemical  reaction  occurs  between  this  organic 
fluid  and  the  suspension. 

The  disseminating  action  of  the  citrate  is  due  to  the  fact  that  the 
suspension  adsorbs  it.  We  find,  indeed,  that  on  treating  a  weak 
solution  of  citrate  with  barium  sulphate,  the  property  of  dissemin- 
ating fresh  barium  sulphate  is  removed.  And,  what  is  more,  if  a 
uniform  amount  of  barium  sulphate  is  added  to  increasing  doses  of 
citrate,  the  absolute  amounts  of  citrate  fixed,  increase,  whereas 
the  proportion  of  the  citrate  adsorbed  varies  relatively  with  the 
initial  mass  of  the  salt. 

*  Bull,  de  le  Cl.  des  Sciences  Acad.  Roy.  de  Belg.,  1907,  No.  6. 


STUDY  OF  MOLECULAR  ADHESION.  425 

A  complex  is  formed  between  the  suspension  and  the  citrate 
similar  to  that  which  occurs  when  barium  sulphate  is  added  to  a 
colloidal  solution  (Section  I).  The  dissemination  of  barium  sul- 
phate by  sodium  citrate  corresponds  with  Spiros'  *  idea.  This 
author  thinks  that  the  presence  of  electrolytes  in  small  amount 
is  necessary  for  the  conservation  of  colloidal  solutions.  The 
salts  capable  of  producing  this  effect  would  vary  with  the  colloid 
under  consideration;  small  amounts  of  alkali  will  sustain  silicic 
acid  and  stannic  acid  in  a  colloidal  condition;  small  amounts  of 
acids  act  in  the  same  manner  on  colloid  ferric  hydrate  and  on 
gelatin.  Malfitano  t  has  shown,  also,  that  colloidal  ferric  hydrate 
filtered  through  collodium  becomes  more  and  more  unstable  as  it 
loses,  its  electrolyte  (HC1) ;  this  writer  believes  that  colloidal  ferric 
hydrate  formed  by  hydrolysis  of  a  ferric  salt  is  unable  to  remain 
alone  in  colloidal  condition,  and  acquires  such  a  condition  only 
through  adsorption  of  the  ions,  Fe  or  H. 

My  own  data  may  be  compared  with  the  facts  that  were  mentioned 
by  Arthus  a  few  years  ago.J  This  writer  found  that  if  sodium 
citrate  (0.5  to  1  per  cent)  is  added  to  an  emulsion  of  clay  in  water, 
the  precipitating  dose  of  the  salts  of  alkalis  or  of  alkali  earths  is 
considerably  increased  (20  to  28  times).  "  Although  in  the  instance 
of  calcium  salts  we  may  advance  the  hypothesis  of  a  double  inter- 
change of  salts  leading  to  the  production  of  calcium  citrate  and 
sodium  chloride,  no  such  hypothesis  could  be  offered  in  the  case  of 
NaCl.  In  this  instance  sodium  citrate  evidences  an  antagonism 
to  sodium  chloride,  the  precipitating  salt;  the  citrate,  then,  may  be 
regarded  as  endowed  with  a  direct  antiprecipitating  property." 
This  phenomenon  is  interesting  when  we  consider  the  inhibiting 
action  of  the  citrate  on  the  coagulation  of  blood  and  milk,  which  is 
not  accompanied  by  precipitation  of  calcium.  Our  researches 
show  that  the  antiflocculating  property  of  sodium  citrate,  observed 
by  Arthus,  is  not  due  to  some  unknown  obstacle  to  flocculation  by 
sodium  salts  in  general  which  this  substance  offers;  we  have  indeed 
met  with  certain  examples  of  flocculation  by  sodium  chloride  which 
are  not  inhibited  by  the  citrate  (in  particular,  flocculation  of  the 

*  Hoffmeister's  Beitrage  zur  chem.  Phys.  u.  Pathologie,  Vol.  5. 
t  C.  R.  Acad.  de  Sciences  de  Paris,  1905. 
}  C.  R.  de  Soc.  de  Biol.,  1902. 


426  STUDIES   IN  IMMUNITY. 

iodide  of  starch).  As  a  result  of  our  experiments  we  conclude 
that  the  action  of  the  citrate  is  directly  on  the  flocculable  sub- 
stance itself  and  due  to  its  adsorption  by  this  substance. 

The  complex,  barium  sulphate  plus  citrate,  can  be  washed  in 
distilled  water  and  resuspended  in  distilled  water  in  the  same  way 
as  the  complex  of  barium  sulphate  plus  a  colloid ;  it  remains  in  sus- 
pension. The  stability  of  this  complex,  however,  is  far  from  being 
as  great  as  complexes  of  barium  sulphate  with  colloids,  and  we  find 
that  it  must  be  washed  with  rather  small  volumes  of  water,  since 
large  amounts  of  water  finally  break  it  up  and  liberate  the  powder. 
The  stability  of  the  complex  is,  however,  sufficient  to  allow  us  to 
study  it  by  subjecting  it  to  various  influences. 

When  we  submit  a  complex  of  barium  sulphate  plus  citrate  that 
has  been  washed  and  suspended  in  distilled  water  to  the  action  of 
an  electric  current  we  find  that  it  is  directed  feebly  though  dis- 
tinctly toward  the  anode.  This  fact,  which  is  much  more  distinct 
with  a  complex  of  barium  sulphate  and  colloid,  shows  that  the 
adsorption  of  an  electrolyte  may  endow  a  suspension  with  a  dis- 
tinct electric  charge.  In  this  connection  we  may  recall  that 
Lottermoser*  has  shown  that  colloidal  solutions  of  Agl  may  be 
obtained  from  KI  and  AgN03  by  means  of  an  electric  current 
which  moves  it  either  toward  the  negative  or  positive  pole,  depend- 
ing on  whether  the  initial  mixture  contains  an  excess  of  KI  or 
an  excess  of  AgNo3.  The  ions,  Ag  or  I,  in  excess,  bring  about 
a  colloidal  state  under  these  conditions  (Solbildner,  de  Jordis),  and 
endow  the  particles  of  Agl  with  a  positive  or  negative  electric 
charge. 

Arthus,  in  a  brief  note  on  the  study  of  sodium  citrate,  shows  not 
only  that  it  inhibits  flocculation  of  clay  by  an  amount  of  NaCl  that 
is  sufficient  ordinarily  to  produce  agglutination,  but  he  also  shows 
that  this  action  of  the  citrate  may  be  overcome  by  a  larger  amount 
of  NaCl.  We  have  subjected  the  complex,  barium  sulphate  plus 
citrate,  to  the  action  of  electrolytes  in  the  same  way  that  we  treated 
the  complex  of  barium  sulphate  plus  colloid.  As  in  this  latter  in- 
stance, the  complex,  barium  sulphate  and  citrate,  is  flocculated 
by  a  salt  and  sediments  rapidly  on  the  addition  of  a  neutral  salt, 
a  base  or  an  acid. 

*  Journ.  fur  prakt.  Chem.  1906,  LXXIII. 


STUDY  OF  MOLECULAR  ADHESION.  427 

We  found  that  the  flocculation  of  the  complex,  barium  sulphate 
plus  gum,  by  a  salt,  is  reversible,  that  is  to  say,  on  the  removal  of 
the  saline  solution  and  suspension  in  distilled  water,  the  dissemin- 
ated condition  comes  back.  In  the  same  way  the  complex,  barium 
sulphate  plus  citrate,  flocculated  by  a  salt,  and  after  the  removal  of 
this  salt  suspended  in  distilled  water,  becomes  disseminated  again. 
There  is  then  a  distinct  analogy  between  the  complexes  of  barium 
sulphate  plus  colloid  and  the  complex,  barium  sulphate  plus  citrate, 
in  respect  to  flocculation  by  electrolytes.  A  reversibility  of  the 
flocculation  of  the  citrate  combination  by  means  of  a  salt  also 
occurs  —  at  least  within  the  limits  of  saline  concentration  which 
we  have  employed  —  on  agglutinating  the  complexes  by  a 
soluble  calcium  salt  in  place  of  the  sodium  salt;  the  complex  after 
the  excess  of  calcium  solution  is  removed  when  suspended  in  distilled 
water  recovers  its  normal  appearance.  We  have  also  noted  that 
if  barium  sulphate,  NaCl,  and  sodium  citrate  are  mixed  together  all 
at  once,  that  the  complex  of  barium  sulphate  plus  citrate  still  forms; 
it  is  quite  as  readily  flocculated  by  the  sodium  chloride  present, 
but  when  washed  and  resuspended  in  distilled  water  it  acts  as  if  it 
had  been  formed  without  the  presence  of  sodium  chloride. 

*** 

To  sum  up,  sodium  citrate  forms  a  complex  with  barium  sulphate 
as  with  other  insoluble  substances  which  causes  a  fragmentation  of 
the  granules  of  the  suspension  into  extremely  small  suspended  par- 
ticles; this  results  in  the  formation  of  a  much  more  delicate  and 
stable  suspension  of  the  sulphate.  The  same  thing  is  true  with  clay. 
Arthus  has,  indeed,  noted  that  the  flocculation  of  clay  by  a  sodium 
salt  requires  much  more  salt  when  citrate  is  present  than  when  it 
is  absent.  The  inhibition  which  citrate  causes  on  the  flocculation 
of  a  suspended  substance  in  water  by  means  of  a  salt  may  be  made 
use  of  in  detecting  the  formation  of  a  complex  between  such  a 
suspension  and  citrate.  We  may  use  this  fact  to  advantage  in  deal- 
ing with  a  material  which  usually  gives  so  fine  a  suspension  in  water 
that  the  addition  of  citrate  produces  no  visible  modification. 
Such  is  the  case  with  calcium  fluoride  (fine  suspensions  of  calcium 
fluoride  in  water  which  are  almost  colloidal  may  be  obtained), 
and  particularly  with  mastic.  Both  these  substances,  which  are 


428  STUDIES   IN  IMMUNITY. 

flocculated  on  the  addition  of  a  sodium  salt,  precipitate  with 
much  greater  difficulty  when  citrate  is  present.  This  fact  may  be 
regarded  as  a  result  of  the  formation  of  complexes  between  the 
calcium  fluoride  or  the  mastic  and  citrate. 

We  may  now  consider  the  appearance  of  an  insoluble  substance, 
the  particles  of  which  undergo  a  much  more  marked  dissemination 
than  does  barium  sulphate  when  citrate  is  added.  Inasmuch  as  the 
particles  of  such  substances  are  extremely  small,  they  may  be  more 
like  the  component  particles  of  a  colloidal  solution. 

We  have  known  for  some  time  that  citrates  of  alkalis,  particularly 
the  insoluble  citrates  of  calcium  salts  " dissolve"  certain  substances 
which  are  insoluble  in  water,  it  is  in  addition  noted  that  the 
citrates  of  alkalis  prevent  precipitation  of  a  copper,  aluminium, 
or  iron  salt  by  a  base.  We  think  that  this  action  of  citrates  of 
alkalis  may  be  comparable  to  the  action  of  sodium  citrate  and 
stable  colloids  on  barium  sulphate ;  it  may  be  that  in  these  cases  the 
citrate  forms  a  complex  with  the  precipitate  of  hydrate  as  soon 
as  it  appears,  so  that  the  hydrate,  instead  of  being  flocculated, 
remains  in  colloidal  solution. 

We  place  a  given  amount  of  well-washed  aluminium  in  increas- 
ingly concentrated  solutions  of  citrate,  and  find,  in  a  few  moments, 
that  cloudiness,  caused  by  the  aluminium,  becomes  more  marked  in 
certain  tubes  and  that  it  then  decreases  and  the  fluid  eventually 
becomes  perfectly  clear.  We  know  that  we  are  not  dealing  with 
an  ordinary  dissolution  phenomenon,  inasmuch  as  the  hydrate 
gives  a  more  marked  cloud  with  the  citrate  than  does  ordinary 
aluminium,  which  is  not  the  case  with  ordinary  dissolutions.  And 
what  is  more,  no  soluble  citrate  of  aluminium  has  been  described  so 
far  as  we  know.*  But  the  reason  for  comparing  a  dissolution  of 
aluminium  with  a  suspension  of  barium  sulphate  in  citrate  is  the 
corresponding  action  of  such  salts  as  sodium  chloride  in  both  in- 
stances. We  have  already  noted  that  the  complex,  barium 
sulphate  plus  citrate,  is  flocculated  on  the  addition  of  sodium 
chloride,  in  the  same  way  we  find  the  "solution"  of  aluminium 
in  citrate  becomes  troubled  when  sufficient  amounts  of  sodium 
chloride  are  added. 

*  In  Wurtz'  "Dictionary  of  Chemistry,"  only  an  insoluble  citrate  of  alu- 
minium transferred  by  an  acid  into  a  very  soluble  gummy  product  is  mentioned. 


STUDY  OF   MOLECULAR  ADHESION  429 

The  opacity  produced  by  sodium  chloride  may  be  due  to  a  double 
decomposition:  the  sodium  citrate  could  not  give  the  insoluble 
substance,  aluminium,  with  sodium  citrate  if  it  reacted  chemically 
with  sodium  chloride,  but  could  give  only  soluble  substances.  We 
must  then  be  dealing  with  a  flocculation  of  very  small  particles 
in  limpid  colloidal  solution  by  sodium  citrate;  these  particles  must 
belong  to  the  complex,  aluminium  plus  citrate.  This  complex,  then, 
would  be  analogous  to  the  complex  of  barium  sulphate  plus  citrate, 
but  instead  of  being  a  fine  suspension,  as  this  complex,  it  would 
appear  in  water  as  an  apparently  colloidal  limpid  solution.  Such 
a  mechanism  probably  accounts  for  the  inhibition  to  precipitation 
of  aluminium  salts  by  bases  which  citrate  gives. 

Notwithstanding,  the  reasons  which  seem  to  favor  this  opinion, 
we  give  this  interpretation  as  an  hypothesis  only.  The  subject 
should  be  studied  more  carefully  from  a  purely  chemical  stand- 
point. 

We  should  like,  however,  to  mention  one  more  fact  which  is  re- 
lated to  the  phenomenon  that  we  have  just  described.  We  know 
that  the  salts  of  aluminium,  for  example  the  sulphate,  are  coagu- 
lants and  agglutinants.  Alum  flocks  certain  dyes,  such  as  fuchsin, 
with  ease.  We  have  found  that  such  a  flocculation  is  completely 
inhibited  by  sodium  citrate.  This  effect  cannot  be  due  to  a  pre- 
cipitation of  the  agglutinating  element  from  the  solution,  for  we 
find  that  these  salts  show  no  precipitation  after  several  days.  It 
may  be  that  the  obstacle  to  the  flocculation  by  alum  produced  by . 
citrate  is  due  to  the  dissemination  of  alum  hydrate,  which  would 
remove  its  precipitating  properties.  This,  to  be  sure,  is  only  a 
hypothesis,  the  value  of  which  must  be  determined  only  by  more 
extensive  experiments. 

* 


# 


The  inhibiting  power  of  sodium  citrate  on  the  flocculation  of 
certain  substances  by  salts  (Arthus)  is  better  understood  when  we 
consider  its  disseminating  effect  on  substances  or  elements  which 
sediment  spontaneously.  In  the  same  way  as  the  suspension  of 
insoluble  substances  by  stable  colloids  is  to  be  considered  as  the 
result  of  the  formation  of  a  complex  between  the  powder  and  the 
colloid,  so  the  disseminating  action  of  sodium  citrate  on  certain 


430  STUDIES  IN  IMMUNITY. 

chemical  precipitates  is  due  to  the  production  of  a  complex  between 
the  precipitate  and  the  citrate.  It  is  probable  that  the  adsorbed 
citrate  diminishes  the  surface  tension  between  the  particles  of  the 
adsorbed  body  and  the  surrounding  fluid ;  it  is  owing  to  this  fact, 
probably,  that  the  modification  brought  about  by  the  citrate  in  the 
physical  condition  of  the  adsorbing  substance  is  similar  to  the  in- 
hibition by  this  salt  to  flocculation  of  adsorbing  substances  like 
clay  or  calcium  fluoride  by  electrolytes.  The  inhibiting  action  of 
citrate  on  adhesion  of  particles  of  the  adsorbing  substance  to  other 
elements  in  colloidal  solution,  or  in  suspension,  is  also  apparently 
due  to  the  same  fact.  Citrate  that  has  been  adsorbed  by  barium 
sulphate  can  oppose,  not  only  the  reciprocal  adhesion  of  the  parti- 
cles of  this  suspension,  but  also  the  adhesion  between  them  and 
stable  colloids  of  other  substances  in  the  same  way  as  a  stable  colloid, 
A  (serum),  is  able  to  inhibit  the  adsorption  of  another  colloid,  B 
(starch  or  gum).  This  is  the  way  that  citrate  prevents  the  adsorp- 
tion of  gum  arabic,  mucin  or  of  starch  by  barium  sulphate;  it  like- 
wise inhibits  the  adhesion  and  subsequent  flocculation  of  fresh 
barium  sulphate  plus  colloid.  In  like  manner,  the  citrate  prevents 
the  adhesion  and  subsequent  flocculation  of  calcium  fluoride  with 
indigo  carmin;  and  finally,  in  a  similar  manner,  it  prevents,  to  a 
certain  extent,  the  adsorption  of  eosin  by  animal  charcoal. 

These  various  examples  suffice  to  show  that  the  adsorption  of 
citrate  by  calcium  fluoride,  barium  sulphate,  animal  charcoal  and 
the  like  may  take  the  place  of  the  adsorption  of  other  substances  by 
these  insoluble  bodies  as  well  as  the  mutual  adhesion  of  the  individ- 
ual particles  of  each  one  of  these  materials.  The  phenomenon  is 
evidently  due  to  the  fact  that  the  affinity  of  a  given  suspension  for 
various  substances  (dyes,  colloids  and  citrate)  varies,  and  also  to 
the  fact  that  this  suspension,  which  is  attracted  at  the  same  time  by 
two  affinities  of  different  intensity,  naturally  yields  to  the  stronger 
one  —  in  our  experiments  to  its  affinity  for  the  citrate.  The  inhibit- 
ing effects  of  citrate  are,  then,  simply  the  result  of  a  struggle  of 
affinities  for  barium  sulphate  between  the  citrate  and  the  other 
substances,  the  adsorption  of  which,  by  the  suspension,  this  citrate 
prevents.  It  is  this  principle  of  a  struggle  between  two  adsorption 
phenomena  which  we  have  employed  in  our  study  on  the  agglutina- 
tion and  dissolution  of  red  blood  cells  by  various  substances. 


STUDY  OF  MOLECULAR  ADHESION.  431 

III. 

The  two  preceding  chapters  have  been  given  over  to  the  consid- 
eration of  adsorption  phenomena  which  occur  between  substances 
neither  of  which  is  cellular,  properly  speaking.  Certain  of  them, 
such  as  barium  sulphate,  calcium  fluoride  and  the  like,  are  insoluble 
inorganic  substances;  and  others  are  stable  colloids  (gum  and  the 
like)  or  electrolytes  (sodium  citrate). 

In  the  mutual  adhesion  between  these  substances  which  we  have 
considered,  one  of  them,  the  colloid,  may  be  replaced  by  a  cellular 
element.  As  we  have  previously  shown,*  barium  sulphate,  when 
mixed  with  red  blood  cells  that  have  been  washed  free  of  all  serum 
and  suspended  in  salt  solution,  adheres  to  the  corpuscles  and  pro- 
duces agglutination  in  large  clumps  composed  of  suspension  and 
corpuscles.  The  agglutination  is  followed  by  dissolution  of  the 
corpuscles  when  sufficiently  large  doses  of  barium  sulphate  are  used. 

We  have  here  an  adhesion  phenomenon  between  two  suspended 
substances  which  may  be  studied  by  subjecting  it  to  the  various 
influences  which  we  have  employed  in  dealing  with  other  adhesion 
phenomena,  as  the  one  between  barium  sulphate  and  starch. 
We  find  here,  too,  that  the  agglutination  of  barium  sulphate  with 
corpuscles  does  not  occur  when  a  stable  colloid  like  serum,  or  when 
sodium  citrate,  is  present,  any  more  than  it  does  in  the  adhesion 
of  barium  sulphate  with  starch.  We  know  from  what  we  have  seen 
in  the  preceding  chapters  what  takes  place  under  these  conditions ; 
it  may  be  that  another  complex,  namely,  barium  sulphate  plus 
serum  or  barium  sulphate  plus  citrate,  takes  the  place  of  the  com- 
plex, barium  sulphate  plus  corpuscles,  which  occurs  under  normal 
conditions ;  in  other  words,  one  phenomenon  of  adhesion  takes  the 
part  of  another  similar  phenomenon. 

We  have  already  demonstrated  that  the  particles  of  the  complex, 
barium  sulphate  plus  citrate,  which  remain  separate  in  the  solution 
in  which  they  have  been  formed,  without  any  tendency  toward 
mutual  adhesion,  are  agglutinated  on  the  addition  of  a  salt  such  as 

*  Gengou,  C.  R.  Acad.  Sciences,  Paris,  1904.  Landsteiner  and  Jagic  noted 
the  effect  of  colloidal  silicic  acid  on  corpuscles  some  time  before  (Wein.  klin. 
Wochenschr.,  1904,  No.  3).  Shortly  after  our  communication,  Madam  Girard- 
Mangan  and  v.  Henri  (C.  R.  Soc.  Biol.,  1904)  in  turn  studied  this  phenomenon 
very  extensively  with  various  colloids. 


432  STUDIES  IN   IMMUNITY. 

NaCl  or  CaCl2.  This  salt  increases  the  affinity  of  the  particles  in 
the  complex  for  one  another.  In  like  manner,  the  agglutination  of 
corpuscles  with  suspensions  may  be  forced  to  appear  even  in  a  citrate 
medium  on  the  introduction  of  sufficient  amounts  of  CaCl2;  and, 
what  is  more,  if  we  use  a  solution  of  7  per  cent  saccharose  instead  of 
0.85  per  cent  salt  solution  as  a  medium,  we  may  produce  an  aggluti- 
nation of  barium  sulphate  with  corpuscles,  which  is  prevented  by  the 
citrate,  on  the  simple  addition  of  NaCl. 

These  experiments  may  all  be  repeated  with  other  insoluble  sub- 
stances, as,  for  example,  CaFl2  and  mastic.  Both  these  substances 
agglutinate  washed  red  blood  cells;  the  agglutination  is  prevented 
either  by  stable  colloids  or  by  sodium  citrate,  and  this  failure  in 
agglutination  is  due  to  the  fact  that  the  adhesion  of  the  insoluble 
substance  with  the  corpuscles  fails  to  occur.  In  the  place  of  the 
complexes,  CaFl2  plus  corpuscles,  or  mastic  plus  corpuscles,  the 
complexes,  CaFl2  plus  stable  colloid  or  mastic  plus  citrate,  occur. 
The  addition  of  a  salt  like  CaCl2  or  NaCl  causes  the  agglutination, 
which  has  been  inhibited  by  the  citrate,  to  appear,  just  as  it  provokes 
the  agglutination  of  the  corpuscles  by  barium  sulphate  in  a  citrate 
medium.* 

*  We  have  shown  in  Section  II  the  inhibiting  effect  of  sodium  citrate  on  pre- 
cipitation of  fuchsin  by  alum.  With  every  reserve  as  to  the  interpretation  of  this 
fact  we  have,  nevertheless,  insisted  on  the  analogy  which  seems  to  exist  between 
the  function  of  the  citrate  in  this  instance  and  its  action  with  suspensions.  It  is 
with  a  similar  reserve  that  we  mention  the  effect  of  the  citrate  on  otlver  pheno- 
mena of  agglutination  produced  by  alum.  As  we  already  know,  Malvoz  (Annales 
de  1'Institut  Pasteur,  1897)  has  shown  that  various  chemical  substances  aggluti- 
nate emulsions  of  bacteria.  Alum  agglutinates  typhoid  bacilli  well;  we  have  also 
found  that  it  agglutinates  red  blood  cells  and  even  dissolves  them  when  a  sufficient 
dose  is  employed.  All  these  phenomena  fail  to  occur  when  even  a  small  amount 
of  sodium  citrate  is  present.  Not  only  does  agglutination  fail  to  appear,  but  we 
have  been  able  to  show  that  the  citrate  prevents  the  adsorption  of  the  alum  by 
the  bacteria  or  the  red  blood  cells,  which  normally  occurs  when  citrate  is  not  pre- 
sent. We  have  also  found  that  bacteria  that  have  been  agglutinated  by  moderate 
doses  of  alum,  and  then  washed  in  distilled  water,  are  well  agglutinated  on  the 
addition  of  NaCl  (this  fact  may  be  compared  with  the  agglutination  of  bacteria 
by  specific  sera  and  with  the  observations  of  Beckhold  (Zeitschrift  fiir  phys. 
Chem.,  Vol.  48)  on  the  flocculation  of  bacteria  by  the  salts  of  heavy  metals).  If 
we  treat  bacteria  which  have  been  mixed  with  alum  in  a  citrated  medium  in  the 
same  way,  the  subsequent  agglutination  by  NaCl  fails  to  occur.  It  is  evident, 
then,  that  the  mode  of  action  of  the  citrate  in  this  instance  seems  comparable  to  its 
action  on  substances  in  aqueous  suspensions  (barium  sulphate,  calcium  fluoride  or 
mastic);  but,  owing  to  the  fact  that  chemical  reactions  may  take  place  between 
alum  and  citrate,  and  thus  prevent  the  manifestations  produced  by  the  alum,  we 
think  it  better  to  reserve  any  interpretation  of  this  fact. 


STUDY  OF  MOLECULAR  ADHESION.  433 

It  should  be  noted,  however,  that  the  agglutination  of  corpuscles 
with  insoluble  bodies  like  barium  sulphate  by  means  of  salts  in  a 
citrated  medium  is  never  so  intense  as  when  there  is  no  citrate 
present.  We  think  this  is  owing  to  the  fact  that  the  salts  do  not 
break  up  the  complex  of  barium  sulphate  and  thereby  restore  the 
suspension  to  its  original  condition,  but  endow  the  very  particles 
of  this  complex  with  a  certain  amount  of  adhesive  property.  We 
have  already  seen  that  flocculation  of  the  complex,  barium  sul- 
phate plus  citrate,  by  a  salt  is  reversible,  that  is  to  say,  that  the  com- 
plex recovers  its  normal  appearance  when  the  flocculating  salt  is 
removed.  The  salt,  then,  endows  the  particles  of  the  complex 
with  a  mutual  adhesive  affinity  (flocculation  of  the  complex),  or 
else  with  an  adhesive  affinity  for  other  suspended  substances 
(corpuscles).  And,  consequently,  the  agglutination  of  corpuscles 
with  an  insoluble  substance  in  a  citrated  medium  when  a  salt  is 
present  would  naturally  not  be  as  great  as  the  one  produced  by  the 
same  amount  of  salt  when  citrate  is  absent;  this,  indeed,  is  what 
we  find  to  be  true.  The  contrary  would  occur  if  the  activating 
salt  broke  up  the  complex  and  restored  the  suspension  to  its  normal 
condition. 

The  action  of  citrate  on  calcium  fluoride,  mastic  and  the  like,  as 
we  have  noted  in  Section  II,  is  not  immediately  perceptible,  as  is  the 
dissemination  of  barium  sulphate.  Citrate  does,  however,  affect 
these  various  substances  in  a  similar  manner  and  prevents  their  ad- 
hesion to  other  substances,  such  as  red  blood  cells,  as  we  have 
already  indicated.  Calcium  fluoride,  an  inorganic  substance,  is  in- 
termediary between  barium  sulphate,  an  inorganic  substance 
that  is  disseminated  by  a  citrate,  and  mastic,  which  is  an  organic 
substance  that  shows  no  immediate  effects  with  citrate.  From  a  con- 
sideration of  mastic  we  may  go  on  to  the  study  of  other  organic 
bodies  which  in  turn  would  seem  to  show  no  reaction  to  the  citrate, 
but  which  are,  however,  influenced  by  it  in  a  similar  manner  to 
calcium  fluoride  and  mastic. 


* 
*  * 


We  find  that  we  can  continue  our  comparison  of  the  adsorption 
phenomena  that  we  have  studied  with  similar  phenomena  in  hemoly- 
sis  produced  by  biological  agents.  We  find  that  citrate  of  sodium 
inhibits  hemolysis  by  eel  serum,  venom,  lecithid  and  alexin  in  the 


434  STUDIES  IN  IMMUNITY. 

same  way  as  it  prevents  the  adhesion  of  the  particles  of  barium 
sulphate  to  one  another  or  to  suspended  substances  like  corpuscles 
or  to  colloidal  substances  like  gum  arabic.* 

It  is  evident  that  this  inhibition  of  hemolysis  by  citrate  is  not  due 
to  a  destruction  of  the  biological  hemolysins  by  this  salt,  inasmuch 
as  we  find  they  can  be  reactivated  by  the  addition  of  an  electrolyte. 
In  the  same  way  that  we  increased  the  adhesive  properties  of 
citrated  barium  sulphate  on  the  addition  of  sodium  chloride  and 
calcium  chloride  so  that  the  particles  of  the  complex  are  able  to 
adhere  to  one  another  and  flock  out,  or  to  adhere  to  other  elements 
and  agglutinate  them,  so,  on  adding  calcium  chloride,  we  are  able 
to  reactivate  the  biological  hemolysins  in  a  citrated  medium.  In 
certain  instances,  as,  for  example,  with  venom,  this  reactivation  may 
be  produced  by  NaCl.  If  the  hemolysis  of  guinea-pig  corpuscles 
by  cobra  venom  is  prevented  in  a  sugar  medium  by  a  sufficient 
amount  of  citrate,  the  inhibition  may  be  removed  by  adding  a  small 
amount  of  sodium  chlorid  to  the  mixture. 

It  might  well  be,  however,  that  citrate  prevents  the  action  of  the 
biological  hemolysins  by  neutralizing  the  calcium  salts  which  are 
necessary  for  their  action.  Such  an  interpretation  would  seem  all 
the  more  admissible  inasmuch  as  with  certain  of  these  hemolysins 
(venom,  lecithid  and  alexin)  the  dissolution  of  the  corpuscles  is 
also  lacking  when  oxalate  or  fluoride  of  sodium  is  added  to  the 
mixture.  Inasmuch  as  it  is  generally  supposed  that  the  inhibition 
of  coagulation  of  the  blood  and  of  milk,  produced  by  these  salts,  is 
due  to  a  neutralization  of  the  calcium  salts,  it  may  well  be  supposed 
that  the  similar  inhibition  of  hemolysis  is  due  to  the  same 
reason. 

Such  a  hypothesis,  however,  is  not  justified.  In  the  first  place 
we  have  to  note  that  although  citrate  of  sodium  prevents  hemolysis 
by  eel  serum,  oxalate  produces  no  such  effect  even  when  large 
amounts  of  iso tonic  oxalate  solution  are  employed.  It  is  therefore 
probable  that  the  mechanism  of  the  inhibition  of  hemolysis  by 
the  citrate  is  similar  in  the  various  other  instances  by  biological 
hemolysins  which  have  been  mentioned. 

From  the  facts  which  follow,  and  for  other  reasons,  we  are  led  to 

*  Bordet  and  Gay,  see  page  403,  have  also  described  similar  facts  in  dealing 
with  the  inactivation  of  alexin  by  sodium  citrate. 


STUDY  OF  MOLECULAR  ADHESION.  435 

believe  that  the  inhibiting  action  is  not  due  to  a  decalcifying  power 
of  the  citrate,  the  oxalate  or  the  fluoride.  If  it  were  so,  we  should 
expect  to  find  a  distinct  parallelism  between  the  intensity  of  the 
anticalcium  property  of  the  citrate  or  oxalate  and  their  antihemo- 
lytic  property.  Such,  however,  is  not  the  case.  Sabatani  (Archiv. 
Ital.  de  Biol.,  1901,  Vol.  36)  has  shown  that  if  we  regard  the  anti- 
calcium  power  of  the  oxalate  as  1,  the  similar  power  in  the  citrate 
may  be  represented  by  0.45.  According  to  this  author  the  anti- 
coagulating  property  of  each  of  these  salts  is  exactly  parallel  to 
their  anticalcium  property.  This,  however,  is  not  true  as  regards 
their  antihemolytic  effect.  We  have  found,  indeed,  that  if  we 
represent  the  antihemolytic  action  of  the  oxalate  as  1,  the 
corresponding  action  of  the  citrate  would  correspond  to  3  as 
regards  hemolysis  by  venom,  to  1.45  in  hemolysis  with  lecethid, 
and  to  1.5  with  alexin  hemolysis. 

We  have  found  that  if  amounts  of  oxalate  which  are  entirely 
sufficient  to  decalcify  the  serum  are  added  to  undiluted  alexin  or  to 
concentrated  venom,  and  allowed  to  remain  with  them  for  24  hours, 
on  removal  of  the  calcium  oxalate  that  has  been  formed,  these 
mixtures,  when  suitably  diluted,  are  quite  as  hemolytic  as  if  no 
oxalate  had  been  mixed  with  them. 

Inasmuch  as  the  citrate  does  not  act  as  a  decalcifying  agent,  we 
are  forced  to  compare  its  action  on  animal  hemolysins  with  its  action 
on  the  insoluble  substances  which  we  have  previously  studied. 
To  render  the  analogy  complete  it  would  be  necessary,  in  order  to 
produce  an  inactivation  of  the  hemolytic  agents  in  a  citrated 
medium,  that  an  inhibition  to  the  adsorption  of  these  substances 
by  the  corpuscles  should  exist.  This  we  find  to  be  the  case :  if  in  a 
citrated  medium  blood  corpuscles  and  any  one  of  the  hemolysins 
which  we  have  mentioned  are  mixed,  the  supernatant  fluid  subse- 
quently removed  may  be  shown  on  reactivation  with  CaCl2  to 
contain  the  hemolytic  agent 

It  would  seem,  then,  that  sodium  ci-trate  prevents  the  action  of 
eel  serum,  venom,  lecithid  and  alexin  on  red  blood  cells  in  the  same 
way  that  it  prevents  similar  action  by  such  substances  as  barium 
sulphate,  calcium  fluoride  and  mastic.  We  have  seen  that  the 
inhibition  of  hemolysis  by  insoluble  substances  like  barium  sulphate, 
by  means  of  citrate,  is  due  to  the  formation  of  complexes  such  as 


436  STUDIES  IN  IMMUNITY. 

barium  sulphate  plus  citrate,  calcium  fluoride  plus  citrate,  etc.,  in 
place  of  the  complexes,  barium  sulphate  plus  corpuscles,  and  the 
like.  It  is  probable,  then,  that  hemolysis  by  biological  agents  fails, 
'when  citrate  is  present,  because  these  substances  form  unions  with 
the  citrate  similar  to  barium  sulphate  plus  citrate,  instead  of  attack- 
ing the  corpuscles.  We  have  seen  that  the  action  of  salts  like  NaCl 
and  CaCLj  on  the  complex,  barium  sulphate  plus  citrate,  is  rever- 
sible, and  we  have  further  seen  that  it  is  owing  to  this  reversibility 
that  this  complex  is  not  always  so  agglutinating  for  red  blood  cells 
when  the  salt  is  added  as  if  the  suspension  in  the  complex  had 
been  restored  to  its  normal  condition  by  means  of  NaCl  or  CaCl2. 
This  also  we  find  to  be  true  with  biological  hemolysins.  These 
latter,  when  inactivated  by  citrate,  can  be  reactivated  by  CaCl2 
or  even  by  NaCl.  This  reactivation,  however,  does  not  always 
endow  the  mixture  with  as  much  activity  as  the  same  amount  of 
hemolysin  would  have  in  a  non-citrated  medium.  It  would  seem, 
then,  as  if  the  reactivating  salts  simply  increase  the  affinity  of  the 
complex  of  hemolysin  plus  citrate  for  the  corpuscles.  This,  to  be 
sure,  is  the  most  plausible  explanation  of  their  activation  of  such 
complexes  by  NaCl;  it  does  not,  however,  authorize  us  at  the  pres- 
ent time  to  lay  aside  the  possibility  of  a  chemical  neutralization  of 
the  citrate  by  CaC^. 

Apart  from  this  question  of  the  mechanism  of  reactivation  by 
CaCl2,  we  may  say  that  it  would  seem  that  the  inhibiting  effect  of 
citrate  on  hemolysis  by  biological  agents  is  due  to  the  same  mechan- 
ism as  its  influence  on  the  various  phenomena  that  have  been  studied 
in  the  preceding  chapters.  There  is  a  very  clear  parallelism  between 
these  two  classes  of  phenomena.  As  a  result,  we  feel  justified  in 
concluding  from  our  researches  that  the  mode  of  union  of  biological 
hemolytic  agents  with  red  blood  cells  would  seem  to  be  an  adsorption 
phenomenon  which  is  comparable  to  the  one  that  occurs  between  red 
blood  cells  (or  the  other  substances  which  we  have  studied)  and  insol- 
uble substances  like  barium  sulphate  or  colloidal  solutions  like  mastic. 
We  have  described  these  various  phenomena  of  adhesion  between 
red  blood  cells  and  insoluble  substances,  or  biological  hemolysins, 
to  the  present,  as  they  occur  in  solutions  of  isotonic  solutions  of 
NaCl  or  in  blood  serum.  The  majority  of  these  phenomena  will 
also  take  place  in  a  solution  of  saccharose  as  well;  the  hemolysis 


STUDY   OF  MOLECULAR  ADHESION.  437 

by  eel  serum  and  by  alexin,  however,  does  not  occur  under  these 
conditions.  We  may  leave  aside  hemolysis  by  alexin,  inasmuch 
as  it  is  known  to  be  very  much  altered  by  dilution  in  a  medium 
which  is  poor  in  salts.  The  failure  of  hemolysis  by  eel  serum  in 
a  sugar  medium  is  not,  however,  due  to  an  alteration  in  the 
hemolysins,  but  to  deprivation  of  salts.  When  we  dialyze  eel 
serum  against  distilled  water  we  find  the  globulins  which  are  pre- 
cipitated by  this  treatment  contain  no  hemolysin,  but  that  this 
substance  remains  intact  in  the  limpid  portion  of  the  dialyzed 
serum.  We  have  found,  moreover,  that  the  reason  this  hemolysin 
does  not  dissolve  red  blood  cells  in  a  sugar  medium  is  due  to  the 
fact  that  under  such  conditions  it  is  not  adsorbed  by  the  cells. 
The  addition  of  salt  to  such  a  mixture,  however,  brings  about 
adhesion  of  the  hemolysin  to  the  cells,  and  their  dissolution. 
When  we  compare  the  activating  power  of  various  salts,  we 
find  that  they  depend  on  the  kation  and  that  the  salts  of  the 
alkali  earths  are  much  more  potent  than  corresponding  salts 
of  alkali  metals;  the  activating  strength  of  the  salts  increases, 
then,  parallel  to  their  flocculating  action  on  unstable  negative 
colloids. 

In  this  case,  also,  it  would  seem  that  the  electrolytes  increase  the 
adhesive  property  of  the  particles  of  the  hemolysin  in  eel  serum  for 
corpuscles  by  increasing  their  superficial  tension  in  the  same  way 
that  they  increase  the  adhesive  power  of  the  particles  of  an  unsta- 
ble negative  colloid  for  one  another.*  This  evidently  does  not  ex- 
plain the  action  of  the  salts;  we  may,  however,  say  that  such 
salts  increase  the  adhesive  power  of  a  suspended  substance  for  other 
suspended  substances  as  if  they  increased  the  affinity  of  the  particles 
of  such  a  suspended  substance  for  similar  particles.  It  would  seem 
to  us  as  difficult  to  explain  the  adsorption  on  the  addition  of  salts, 
of  substance  A  by  substance  B  —  and  particularly  of  the  hemolysin 
of  eel  serum  by  red  blood  cells  —  as  a  purely  chemical  phenomenon, 
as  it  is  to  explain  the  reciprocal  adhesion  between  particles  of  a 

*  This  phenomenon  may  be  compared  with  the  facts  that  have  been  mentioned 
by  Nasse  (Pfliig.  Archiv.  fur  Physiol.,  1885,  Vol.  37)  and  by  Bayliss  (Biochem. 
Journal,  I) .  Nasse  found  that  the  adsorption  of  iodine  by  glycogen  is  increased 
by  salts;  Bayliss  demonstrated  that  the  coloring  power  of  electronegative  colloidal 
dyes  for  paper  is  increased  by  the  kations  and  inhibited  by  the  anions,  and  that 
the  inverse  is  true  with  electropositive  colloidal  dyes 


438  STUDIES  IN  IMMUNITY. 

given  substance,  when  salts  are  added,  by  the  same  method. 
This  adhesive  power,  which  the  hemolysin  in  eel  serum  has  only 
when  electrolyes  are  present,  would  seem  to  exist  normally  in  other 
substances,  either  for  other  suspended  elements  or  for  other  particles 
of  their  own  suspension. 

This  faculty  of  adhesion  which  is  spontaneous  with  certain  mate- 
rials, and  is  brought  about  only  when  electrolytes  are  present  in 
other  materials,  is  combated  by  the  citrate,  which  substitutes  one 
adhesion  for  the  other  adhesions  which  these  substances  may  show. 
The  action  of  the  citrate,  its  mechanism  and  the  opposing  effect  of 
electrolytes  are  precisely  the  same  whether  we  study  them  in  distinct 
adsorption  phenomena,  such  as  the  adsorption  of  one  substance  in 
colloidal  solution  (gum  arabic),  or  in  suspension  (corpuscles)  with 
an  insoluble  substance  (barium  sulphate),  or  when  we  study  the 
hemolytic  phenomena  produced  by  the  biological  agents  that  have 
been  studied  by  bacteriologists. 

*** 

It  should  be  recalled  in  this  connection  that  Bordet  has  long 
maintained  that  the  neutralization  of  a  toxin  by  an  antitoxin  is 
comparable  to  the  phenomena  of  dyeing  rather  than  to  the  ordinary 
chemical  reactions  expressed  by  equations.  We  cannot  mention 
all  the  facts  that  serve  to  support  this  theory,  in  this  place,  but  we 
may  recall  that  certain  experiments  led  Bordet  to  postulate  that 
the  dose  of  alexin,  fixed  by  a  given  quantity  of  corpuscles,  would 
vary  according  to  the  initial  mass  of  alexin  that  was  present. 
Certain  other  researches  also  led  him  to  the  conclusion  that  a  given 
dose  of  anti-alexin  will  neutralize  variable  amounts  of  alexin  in 
accordance  with  the  manner  in  which  the  two  substances  are  mixed. 
We  meet  with  the  same  fact  in  considering  the  neutralization  of 
certain  toxins  by  their  antitoxins ;  this  fact  is  generally  described  as 
Danysz'  phenomenon. 

It  was  particularly  on  the  basis  of  the  facts  concerned  in  the 
reactions  of  alexin  with  corpuscles  on  the  one  hand  and  with  anti- 
alexin  on  the  other,  that  Bordet  formed  his  theory  of  the  neutral- 
ization of  toxins  by  antitoxins.  It  seems  not  without  interest  to 
compare  this  writer's  conceptions  with  the  conclusions  which  we 
have  arrived  at  in  this  work,  in  so  far  as  concerns  the  mode  of 


STUDY  OF   MOLECULAR  ADHESION.  439 

reaction  of  the  alexin  on  corpuscles.  We  have  also  been  led  to 
compare  this  reaction  with  the  phenomena  of  molecular  adhesion 
but  arrived  at  this  comparison  in  a  somewhat  different  manner. 
Our  conclusions,  we  believe,  may  also  be  considered  as  an  additional 
experimental  support  for  Bordet's  theory. 


XXIII.      THE  PHENOMENA  OF  ADSORPTION  AND  THE 
CONGLUTININ    OF   BOVINE   SERUM  * 

BY  DRS.   J.   BORDET   AND   OSWALD   STRENG. 

One  of  us  has  suggested  and  maintained  for  some  time  the 
opinion  that  the  phenomena  of  union  of  antigens  with  antibodies  in 
serum  belong  rather  in  the  category  of  phenomena  of  molecular 
adhesion  than  among  chemical  reactions,  properly  speaking;  in 
other  words,  they  are  apparently  adsorption  phenomena. 

That  the  modifications  on  certain  cells  brought  about  by  specific 
serum  frequently  resemble  phenomena  which  have  no  relation  to  a 
chemical  reaction  both  in  their  general  appearance  and  in  their 
course,  is  undoubted.  Ten  years  ago  one  of  us  brought  out  the 
fact  that  hemolysis  by  an  active  serum  obtained  by  immunizing 
animals  against  the  blood  of  a  foreign  species  resembles  in  certain 
respects  the  hemolysis  produced  by  distilled  water;  in  both  instances 
the  stromata  persist  and  the  hemoglobin  is  unaltered;  when  oval 
corpuscles  are  affected  they  become  spherical  and  swell  up  before 
losing  their  coloring  matter.  A  still  more  striking  analogy  was 
noted  shortly  afterward:  the  agglutination  of  bacteria  by  normal 
serum  or  specific  serum  takes  place  only  in  presence  of  a  salt;  and 
the  flocculation  which  this  electrolyte  brings  about  in  bacteria 
that  have  been  previously  treated  with  serum  is  very  comparable  to 
the  effect  produced  by  the  same  electrolyte  on  a  suspension  of 
clay  or  colloidal  emulsions  like  mastic.  This  fact  gave  rise  to  the 
idea  that  we  should  doubtless  be  able  to  obtain,  by  immunizing 
animals,  sera  capable  of  flocculating  finely  suspended  organic 
substances,  or  albuminoid  substances  like  casein,  and  led  to  the 
discovery  of  "lactoserum,"  that  is,  the  immune  serum  of  animals 
that  have  been  treated  with  injections  of  milk.f 

*  Les  ph6nom6nes  d'absorption  et  la  conglutinine  du  s£rum  de  bceuf.     Cen- 
tralhlatt  fur  Bakteriologie,  1st  Abt.  Orig.  Vol.  49,  1909,  p.  260. 
t  Bordet,  p.  155. 

440 


THE  PHENOMENA  OF  ADSORPTION.  441 

We  recognize  that  these  facts  were  indicative  simply  and  were 
not  an  aosolute  demonstration.  As  concerns  the  phenomenon  of 
agglutination,  Bordet  noted  that  it  takes  place  in  two  distinct 
phases:  during  the  first  a  complex  is  formed  by  the  union  of  the 
agglutinin  with  the  bacteria;  in  the  second  the  salt  precipitates  this 
complex.  In  the  analogous  instances  of  clay  flocculation  we  have 
to  deal,  then,  with  the  second  phase  only  and  obtain  no  light  on  the 
mode  of  union  of  the  agglutinin  with  the  receptors  of  the  bacteria. 

The  presumptions  that  we  deduced  from  these  preliminary 
observations  led  to  researches  on  molecular  adhesion  which  soon 
brought  out  serious  experimental  confirmations.  In  comparing  the 
fixation  of  hemolysins  on  corpuscles  with  the  adsorption  of  analin 
dye  by  filter  paper,  Bordet  mentioned  as  an  analogy  that  the  cor- 
puscles do  not  fix  the  active  substances  according  to  the  law  of  defi- 
nite proportions.  Thus  we  find  that  a  given  volume  of  hemolytic 
serum  destroys  a  larger  amount  of  corpuscles  when  they  are  added 
to  it  in  a  single  dose  than  when  they  are  added  in  successive  divided 
doses  at  relatively  long  intervals.  If  the  second  method  is  used  it 
is  evident  that  the  first  corpuscles  become  overladen  with  active 
principles  and  monopolize  a  larger  amount  than  is  necessary  to 
hemolyze  thein,  thus  leaving  less  for  subsequently  added  corpuscles. 
In  the  same  way,  if  a  leaf  of  filter  paper  is  placed  in  a  given  amount 
of  methylene-blue  solution  it  takes  a  uniform  color  of  a  given  inten- 
sity. But  if  a  piece  of  the  same  size  is  first  cut  up  into  small 
pieces  and  these  pieces  introduced  at  intervals,  we  find  that  the 
first  of  them  stain  deeply  and  the  last  ones  find  no  color  left. 
The  same  experiments  were  made  with  the  same  result  by  Danysz 
with  ricin  and  antiricin,  by  Bordet  with  alexin  and  anti-alexin,  and 
by  von  Dungern  with  diphtheria  toxin  and  antitoxin :  the  neutral- 
izing dose  varies  depending  on  whether  the  toxin  is  added  in  a 
single  or  in  several  doses.  The  same  conclusion  may  be  drawn 
with  agglutinins  and  bacteria,  as  Craw  has  shown.  We  do  not 
wish  to  insist  at  this  point  on  the  obvious  interest  brought  out  by 
the  fact  that  toxins  unite  with  antitoxins  in  variable  proportions.* 

The  analogies  between  phenomena  brought  about  by  sera  and 
those  due  to  substances  which  certainly  do  not  act  chemically, 

*  Consult  on  this  subject  the  former  article  on  the  mode  of  action  of  antitoxins 
on  toxins,  p.  259. 


442  STUDIES   IN  IMMUNITY. 

but  by  properties  of  adhesion,  have  been  very  instructive.  The 
agglutination  and  hemolysis  of  red  blood  cells  by  barium  sulphate 
(Gengou),  by  silicic  acid  (Landsteiner  and  Jagic),  and  similar  facts 
brought  out  by  researches  of  many  writers  on  colloids,  gave  greater 
and  greater  importance  to  the  effect  of  adhesion  in  the  reactions 
between  antisera  and  their  antigens. 

In  hemolysis  in  particular,  the  phenomenon  of  alexin  absorption 
by  corpuscles  laden  with  sensitizer  (amboceptor)  has  given  rise  to 
most  active  controversies.  The  sensitizing  theory  of  Bordet  consists 
essentially  in  the  conception  that  the  sensitizer,  which  possesses  in 
itself  no  particular  affinity  for  the  alexin,  forms,  by  uniting  with 
certain  substances  in  the  corpuscle,  a  complex  endov/ed  with  prop- 
erties of  molecular  adhesion  for  the  alexin  which  are  not  possessed 
by  the  normal  corpuscle.  The  corpuscle  that  has  been  modified  in 
this  manner  would  seem  to  acquire  the  property  of  adsorbing  alexin, 
of  manifesting  an  avidity  which  may  be  compared  to  that  which 
occurs  in  the  fixation  of  fibrinogen  or  other  albuminous  substances 
by  chemically  inert  particles  (for  example,  barium  sulphate)  or  to 
the  union  of  diphtheria  toxin  with  precipitates  of  calcium,  etc.  As 
we  know,  this  point  of  view  differs  from  the  amboceptor  theory  of 
Ehrlich,  in  accordance  with  which  this  antibody  owes  its  interme- 
diary function  to  the  fact  that  it  possesses  in  its  molecule  two  com- 
bining atom  groups:  one(complementophilic),  which  unites  with  the 
alexin,  and  the  other  (cytophilic),  which  combines  with  the  corpus- 
cle. We  shall  not  take  up  at  this  point  a  general  discussion  of  these 
theories,  which  one  of  us  in  collaboration  with  Dr.  Gay  has  recently 
considered ;  *  we  may  recall  that  no  experiment  has  ever  shown 
that  sensitizers  can  unite  with  alexin  in  absence  of  antigens.  Such 
a  combination  under  certain  conditions  has  been  asserted  by  Ehrlich 
and  Sachs, t  as  a  result  of  their  researches  on  the  hemolysis  of  guinea- 
pig  corpuscles  by  horse  serum  to  which  bovine  serum  has  been 
added. 

Bordet  and  Gay  t  undertook  two  years  ago  the  study  of  this  in- 
stance of  hemolysis  and  arrived  at  the  conclusion  that  Ehrlich  and 
Sachs'  interpretation  is  inexact.  Their  conclusion,  in  turn,  has 

*  See  p.  398. 

t  See  Studies  on  Immunity,  Ehrlich-Bolduan,  Wiley  &  Co.,  p.  209. 

t  See  p.  363. 


THE  PHENOMENA  OF  ADSORPTION.  443 

been  objected  to  by  Sachs  and  Bauer  *  who  maintain  integrally 
the  opinion  that  was  advanced  by  Ehrlich  and  his  collaborator. 
We  feel  led,  therefore,  to  return  to  this  question,  which  appears  to 
us  to  present  more  interest  than  might  at  first  be  presumed,  on  the 
general  problem  of  the  action  of  sera  and  particularly  on  certain 
of  their  practical  applications. 

We  may  recall  the  facts  briefly.  Ehrlich  and  Sachs  noted  that 
guinea-pig  red  corpuscles  remain  intact  in  fresh  horse  serum,  but 
are  readily  hemolyzed  by  a  mixture  of  this  serum  with  bovine 
serum  that  has  previously  been  heated  to  56  degrees,  which  fact 
gives  rise  to  the  supposition  that  there  is  a  strong  amboceptor  in 
bovine  serum  which  allows  the  corpuscles  to  fix  horse  alexin  and 
be  hemolyzed  by  it.  But  Ehrlich  and  Sachs  found  in  addition  that 
if  the  corpuscles  are  placed  in  contact  with  heated  bovine  serum 
and  subsequently  centrifugalized  to  remove  this  serum,  and  fresh 
horse  serum  is  then  added,  no  hemolysis  appears.  It  would 
seem  as  if  the  bovine  sensitizer  required  the  presence  of  the 
horse  alexin  in  order  to  become  fixed  on  the  corpuscles.  A  fact 
which  seems  to  confirm  this  idea  is  that  heated  bovine  serum  that 
has  been  previously  treated  with  a  large  quantity  of  guinea-pig 
corpuscles  (which  latter  are  then  removed  by  centrifugalizing) 
does  not  act  as  if  it  had  been  deprived  of  this  amboceptor.  It 
is  able,  indeed,  to  form  a  hemolytic  mixture  with  fresh  horse 
serum,  which  destroys  fresh  guinea-pig  corpuscles.  At  least  this  is 
Ehrlich  and  Sachs'  interpretation  of  the  experiment.  This  opinion 
is  translated  into  words  which  conform  better  with  their  theories 
by  saying  that  the  cytophilic  group  of  the  bovine  amboceptor  is 
not  able  to  react  with  the  corpuscle  unless  the  complementophilic 
group  has  previously  satisfied  its  affinity  for  alexin.  The  mutual 
relation  between  corpuscle,  alexin  and  sensitizer  would  seem,  in 
the  present  instance  to  be  unusual  because,  in  dealing  with  the 
majority  of  hemolytic  sera,  we  find  that  the  corpuscles  absorb 
the  sensitizer  without  the  presence  of  alexin. 

Bordet  and  Gay  in  their  study  on  this  subject  were  led  to  quite 
different  conclusions.  Bovine  serum  possesses,  in  their  opinion, 
distinct  properties  which  are,  however,  quite  different  from  those 
noted  by  Ehrlich  and  Sachs.  According  to  Bordet  and  Gay,  there 

*  Arbeiten  ans  dem  K.  Inst.  fur  exper.  Therapie,  1907. 


444  STUDIES   IN  IMMUNITY. 

is  present  in  bovine  serum  a  particular  substance  which  is  neither  an 
amboceptor,  an  agglutinin,  nor  an  alexin,  and  which  consequently 
differs  from  the  active  substances  that  have  been  previously  recog- 
nized in  studies  on  immunity.  This  substance  has  the  property  of 
combining  with  corpuscles  that  have  already  been  laden  with  sen- 
sitizer  and  alexin,  and  this  combination  generally  favors  hemolysis. 

Two  preliminary  ideas  are  necessary  in  order  that  the  facts  may 
be  clearly  understood.  The  first  is  that  horse  alexin  is  very  weak 
in  so  far  as  producing  hemolysis  is  concerned.  Corpuscles  that  have 
been  strongly  sensitized  by  a  heated  immune  serum  show  little  or 
no  hemolysis  on  the  addition  of  horse  serum.*  Consequently, 
when  horse  alexin  is  used,  an  absence  of  hemolysis  does  not  prove 
that  the  corpuscles  concerned  are  not  sensitized,  that  is  to  say, 
have  not  fixed  the  amboceptor. 

The  second  point  is,  that  fresh  horse  serum  contains  a  relatively 
strong  amboceptor  for  guinea-pig  corpuscles  which  produces  a  good 
alexin  fixation.  The  fact,  then,  that  fresh  horse  serum  does  not 
destroy  the  corpuscles  is  not  due  to  its  lack  of  sensitizer,  but  be- 
cause its  alexin  is  incapable  of  hemolyzing.  If  this  alexin  is  replaced 
by  another,  for  example,  if  fresh  guinea-pig  serum  is  added  to  the 
mixture  of  guinea-pig  corpuscles  and  horse  serum,  hemolysis  takes 
place.  We  are  forced,  then,  to  admit  in  Ehrlich  and  Sachs'  experi- 
ment, which  consists  in  mixing  fresh  horse  serum  and  heated  bovine 
serum  with  guinea-pig  corpuscles,  the  presence  of  two  amboceptors, 
bovine  and  horse,  both  of  which  produce  a  sensitization,  but 
either  one  of  which  suffices  to  produce  the  result.  Either  one 
of  these  amboceptors  may  indeed  be  used  alone  without  changing 
the  result  of  the  experiment.  This  fact,  to  be  sure,  is  scarcely  accept- 
able to  Ehrlich  and  Sachs,  according  to  whom  the  amboceptor  in 
their  experiment  is  furnished  entirely  by  the  bovine  serum.  And 
when  these  authors  find  on  treating  bovine  serum  with  guinea-pig 
corpuscles  that  the  serum  still  retains  its  property  of  rendering  horse 
serum  hemolytic  for  new  corpuscles,  their  conclusion  is,  as  we  have 
seen,  that  the  bovine  amboceptor  has  remained  free  in  spite  of 
contact  with  the  corpuscles. 

*  For  example,  bovine  corpuscles  sensitized  by  rabbit  antibovine  immune 
serum.  The  hemolytic  weakness  of  horse  alexin  is  not  the  same  with  all  species 
of  corpuscles. 


THE  PHENOMENA  OF  ADSORPTION.  445 

The  interpretation  of  Bordet  and  Gay,  on  the  contrary,  is  the 
following : 

The  contact  with  the  corpuscles  has  deprived  the  bovine  serum  of 
its  amboceptor;  this  amboceptor,  however,  may  be  replaced  by  the 
sensitizer  in  horse  serum,  which  latter  serum  also  furnishes  the 
alexin.  Under  these  conditions  the  bovine  serum,  which  still  re- 
mains necessary  for  hemolysis,  acts  only  on  account  of  the  peculiar 
substance  which  it  possesses,  which,  as  we  have  just  seen,  is  neither 
amboceptor  nor  alexin,  but  endowed  with  the  peculiar  characteristic 
of  allowing  horse  alexin  to  produce  hemolysis. 

We  may,  for  the  sake  of  clearness,  explain  this  idea  somewhat 
more  fully  before  recalling  the  experiments  which  proved  its 
exactness.  This  particular  substance  in  bovine  serum  which  resists 
heating  to  56  degrees  and  which  for  simplicity  Bordet  and  Gay 
called  "  bo  vine  colloidal  substance"  does  not  react  with  normal 
corpuscles;  it  unites  only  with  corpuscles  that  have  been  laden  both 
with  amboceptor  and  alexin. 

This  union  or  adsorption  of  the  particular  substance  in  question 
by  sensitized  and  alexinized  corpuscles  is  accompanied  by  visible 
manifestations;  the  corpuscles  on  uniting  with  this  substance  are 
energetically  agglutinated  and  become,  with  certain  exceptions 
which  we  shall  later  mention,  more  apt  to  hemolyze.  This  phe- 
nomenon of  agglutination  of  the  corpuscles  into  large  clumps  in 
Ehrlich  and  Sachs'  experiment,  although  extremely  characteristic, 
was  apparently  not  noted  by  these  investigators,  who  were  more 
interested  in  the  hemolysis.  Heated  bovine  serum  alone  aggluti- 
nates guinea-pig  corpuscles  only  feebly;  horse  serum  agglutinates 
them  better,  but  rather  slowly.  A  mixture  of  the  two  sera,  how- 
ever, brings  about  in  a  very  few  moments  an  extremely  marked 
clumping  of  large  masses  of  corpuscles,  which  soon  afterward  lose 
their  hemoglobin. 

Bordet  and  Gay's  explanation  of  this  is  then  quite  evident;  the 
corpuscles  in  such  a  mixture  fix  first  the  bovine  and  horse  ambocep- 
tors  and  then  the  alexin.*  A  certain  amount  of  time  is  necessary  for 
this  to  take  place,  but  as  soon  as  the  corpuscles  become  laden  with 

*  It  would  be  very  difficult  to  define  the  proportions  in  which  each  of  these 
two  sensitizers  is  fixed;  it  is,  moreover,  of  little  importance.  The  essential  fact 
is  to  recognize  that  either  of  them  will  suffice  to  sensitize. 


446  STUDIES  IN  IMMUNITY. 

these  substances  they  are  then  in  a  position  to  unite  with  the  bovine 
colloid,  which  clumps  them  and  renders  them  more  susceptible  to 
hemolysis.  The  second  assertion  of  Bordet  and  Gay,  to  which  we 
have  already  referred,  is  that  bovine  serum  presents  no  abnormality 
so  far  as  its  amboceptor  is  concerned.  This  sensitizer  is  able  to 
unite  with  the  corpuscles  when  the  alexin  is  absent,  contrary  to  the 
opinion  of  Ehrlich  and  Sachs.  Heated  bovine  serum  that  has  been 
subjected  to  a  large  amount  of  guinea-pig  corpuscles  still  acts  as  if  it 
had  lost  no  essential  element  because  its  colloid  has  been  retained. 
This  is  due  to  the  fact  that  the  corpuscles  used  to  absorb  the  serum 
were  not  alexinized  and  consequently  could  not  combine  with  the 
colloid.  Such  a  treated  serum,  however,  has  lost  its  amboceptor. 
But  inasmuch  as  this  lack  may  be  replaced  by  the  sensitizer  in  horse 
serum,  a  mixture  of  bovine  serum  treated  in  this  manner,  of  fresh 
horse  serum  and  of  guinea-pig  corpuscles,  acts  almost  exactly  as 
when  intact  bovine  serum  is  used. 

It  is  necessary  to  explain  these  matters  rather  in  detail,  inasmuch 
as  the  mechanism  in  the  experiment  under  consideration  is  rather 
complex  and  must  be  attentively  considered.  Many  similar 
experiments  have  been  offered  by  Bordet  and  Gay  to  demon- 
strate the  function  of  this  bovine  colloid  substance  clearly.  If 
we  take  washed  bovine  corpuscles  and  add  to  them  a  mixture  of 
fresh  horse  serum  and  heated  bovine  serum,  nothing  occurs.  But 
if,  in  a  similar  experiment,  we  use  sensitized  bovine  corpuscles, 
we  find  that  they  become  violently  agglutinated  and  are  then 
hemolyzed.  This  phenomenon  is  quite  identical  with  the  one  in 
Ehrlich  and  Sachs'  experiment  on  guinea-pig  corpuscles.  In  both 
instances  bovine  serum  is  necessary;  neither  the  bovine  corpuscles 
nor  the  guinea-pig  corpuscles  are  distinctly  altered  by  the  horse 
serum  alone.  In  the  experiment  with  bovine  corpuscles,  however, 
it  cannot  be  asserted  that  the  bovine  serum  acts  as  an  amboceptor. 
It  must  certainly  act  on  account  of  the  particular  colloidal  substance. 
Since  it  is  indispensable  that  such  corpuscles  should  be  sensitized 
and  that  alexin  should  be  present,  the  only  possible  conclusion  to 
be  drawn  is,  that  corpuscles  that  have  been  sensitized  and  alexinized 
are  then  capable  of  uniting  with  bovine  colloid,  which  substance 
brings  about  clumping  and  hemolytic  results.  The  objection  may 
be  raised,  as  indeed  Sachs  and  Bauer  have  done  in  their  article, 


THE  PHENOMENA  OF  ADSORPTION.  447 

that  in  this  experiment  with  bovine  corpuscles  certain  unexpected 
reactions,  such  as  certain  anticomplement  effects,  may  take  place 
between  the  bovine  serum  and  the  horse  serum.  This  objection, 
however,  can  scarcely  be  maintained,  inasmuch  as  the  horse  serum 
may  be  replaced  by  any  other  alexic  serum  without  any  change  in  the 
result.  The  experiment,  indeed,  becomes  simpler  if,  as  Bordet  and 
Gay  have  done,  we  add  simply  fresh  bovine  serum  to  sensitized 
bovine  corpuscles.  Hemolysis  appears,  which  is  not  surprising, 
but  it  is  preceded  —  and  this  is  the  particular  point  to  be  noted  —  by 
an  extremely  energetic  agglutination  which  characterizes  the  union 
of  the  colloidal  substance.  This  phenomenon,  moreover,  is  another 
indication  of  the  phenomenon  that  has  been  previously  noted.  In 
the  last-mentioned  experiment  bovine  serum  acts  in  two  ways: 
both  as  a  complement  and  as  a  colloid.* 

May  the  somewhat  vague  name  of  colloidal  substance  be  still 
employed  to  designate  this  active  substance  in  bovine  serum? 
Bordet  and  Gay  made  use  of  it  only  provisionally.  As  we  know,  this 
colloid  is  absorbed  by  various  corpuscles  provided  they  be  sensitized 
and  alexinized,  and  the  union  is  indicated  by  a  very  evident  phenom- 
enon of  agglutination  in  large  masses.  This  colloidal  substance 
should  not  be  confounded  with  the  ordinary  agglutinins  and  yet 
should  be  designated  with  some  name  that  suggests  the  fact  that  it 
produces  agglutination.  For  this  reason  we  propose,  from  now  on, 
to  give  the  name  of  "  conglutinin "  to  this  substance  in  bovine 
serum,  and  to  refer  to  the  agglutination  which  it  produces  as 
"  conglutination." 

SECTION  I 
SACHS  AND  BAUER'S  OBJECTIONS. 

Bordet  and  Gay  naturally  brought  forward  numerous  proofs,  which 
we  cannot  reproduce  here,  to  support  their  interpretation.  These 
proofs  will  be  found  in  the  previous  article  of  1906.  We  wish  at 
this  time  to  offer  certain  new  arguments,  which,  in  addition  to  the 
previous  ones,  permit  us  to  refute  the  objections  that  have  been 

*  Muir  and  Browning,  Journal  of  Hygiene,  Vol.  6,  page  20,  noted,  indepen- 
dently of  Bordet  and  Gay,  that  rabbit-antibovine  serum  when  added  to  bovine 
blood  produces  a  marked  agglutination  of  the  corpuscles,  in  producing  which  the 
presence  of  alexin  is  necessary. 


448  STUDIES  IN  IMMUNITY. 

offered  by  Sachs  and  Bauer,  and  also  offer  further  information  on 
the  properties  of  the  conglutinin.  / 

No  comparison  between  ordinary  agglutinins  and  the  conglutinin 
is  necessary.  The  two  substances  are  evidently  different.  Agglu- 
tinins have  no  need,  for  their  action,  of  sensitized  and  alexinized 
corpuscles,  as  does  the  conglutinin.  In  the  experiment  that  has 
just  been  mentioned,  that  is,  the  agglutination  of  sensitized  bovine 
corpuscles  by  fresh  bovine  serum,  it  is  evident  that  we  are  not 
dealing  with  an  ordinary  agglutinin.  It  is,  however,  interesting 
to  compare  the  development  of  the  two  phenomena  by  adding 
bovine  serum  to  two  different  species  of  corpuscles,  one  of  which  it 
agglutinates  and  the  other  of  which  it  conglutinates.  Guinea-pig 
and  horse  red  blood  cells,  respectively,  fulfill  these  conditions. 
We  may  consider,  in  the  first  place,  the  agglutinating  effect :  to 
two  tubes  containing  1  c.c.  of  salt  solution  and  0.1  of  a  cubic 
centimeter  of  heated  bovine  serum,  we  add  0.05  of  a  cubic  centimeter 
of  washed  blood  either  from  the  guinea-pig  or  the  horse.  The 
guinea-pig  corpuscles  are  not  markedly  agglutinated,  those  of  the 
horse  are  rapidly  agglutinated ;  in  this  case  we  are  dealing  with  an 
ordinary  agglutinin.  We  may  then  consider  the  conglutinin,  that 
is  to  say,  in  a  similar  experiment  we  may  employ  fresh  bovine  serum 
instead  of  heated  bovine  serum.  The  horse  corpuscles  act  just  as 
they  do  with  heated  serum :  there  is  rapid  agglutination  of  a  similar 
intensity,  but  no  real  conglutination  appears.*  The  guinea-pig 
corpuscles  show  no  immediate  agglutination  any  more  than  with 
the  heated  serum.  After  10  or  15  minutes  conglutination  appears 
and  the  corpuscles  collect  into  large  clumps  which  gradually 
hemolyze.  It  is  to  be  noted  that  although  a  certain  amount  of 
time  is  necessary  for  the  fixation  of  the  alexin,  the  clumping  by 
the  conglutinin  is  much  more  marked  than  that  by  the  ordinary 
agglutinin. 

We  may  now  consider  Sachs  and  Bauer's  objections: 
(a)     These  authors  consider  Bordet  and  Gay's  interpretation  as 
inacceptable  since  it  necessitates  the  admission  of  a  new  substance, 

*  This  is  due  to  the  fact  that  they  do  not  fix  the  alexin  sufficiently,  or,  in 
other  words,  to  the  fact  that  the  bovine  serum  is  not  sufficiently  sensitizing 
for  horse  corpuscles.  As  Streng  has  shown  in  his  memoir  on  the  anticomple- 
ment  (Zeitschrift  fiir  Immunitatsforschung,  vol.  1),  horse  corpuscles  are  perfectly 
well  conglutinated  by  fresh  bovine  serum  when  they  have  previously  been  sen- 
sitized by  a  specific  immune  serum. 


THE  PHENOMENA  OF  ADSORPTION.  449 

the  conglutinin,  in  the  bovine  serum,  which  has  never  been  de- 
scribed in  other  sera.  But  as  we  know,  bovine  serum  is  in  itself 
rather  unusual  and  why  should  not  the  effect  that  it  produces  be 
likewise  so?  Ehrlich  and  Sachs'  theory,  moreover,  is  also  rather 
exceptional  inasmuch  as  it  presupposes  that  a  sensitizer  does  not 
unite  with  the  corpuscles  when  the  alexin  is  absent. 

(b)  Sachs  and  Bauer  seem  to  consider  it  axiomatic  that  the 
properties  of  agglutination  and  hemolysis  cannot  be  attributed  to  a 
single  substance.     Gengou,  as  we  know,  has  noted  that  a  suspension 
of  barium  sulphate  will  agglutinate  and  hemolyze  red  blood  cells. 

(c)  In  their  criticism  of  our  work  our  contradictors  concern  them- 
selves only  with  the  hemolysis.    They  make  no  attempt  to  explain 
the  marked  clumping  of  the  corpuscles  and  do  not  mention  whether 
they  consider  our  interpretation,  at  least  in  so  far  as  this  congluti- 
nation is  concerned,  as  admissible. 

(d)  Sachs  and  Bauer  insist  strongly  on  the  fact  that  heated  bovine 
serum  is  capable  of  sensitizing  guinea-pig  corpuscles.     We  know 
this  fact  and  have  never  denied  it.     We  have  simply  said  that  in 
the  Ehrlich  and  Sachs'  mixture  the  bovine  sensitizer  is  unnecessary 
on  account  of  the  presence  of  a  similar  stronger  substance  in  horse 
serum. 

(e)  The  activity  of  heated  bovine  serum  is  due  to  the  fact,  accord- 
ing to  Bordet  and  Gay,  that  sensitized  and  alexinized  corpuscles 
absorb  the  conglutinin.     For  example,  if  bovine  corpuscles  are  sen- 
sitized with  a  specific  immune  serum  from  the  rabbit,  subsequently 
treated  with  horse  alexin,  and,  after  being  carefully  washed,  heated 
bovine  serum  is  added,  this  latter  serum  loses,  if  not  all,  at  least  part 
of  its  conglutinin  and  becomes  much  less  able  to  form  a  congluti- 
nating  and  hemolytic  mixture  with  horse  alexin  either  for  bovine 
corpuscles  or  for  guinea-pig  corpuscles.    Bordet  and  Gay  have  made 
this  assertion.     The  experiment  is,  however,  denied  by  Sachs  and 
Bauer. 

We  are  dealing  here,  not  with  an  interpretation,  but  with  a  fact, 
and  we  insist  on  its  accuracy.  In  the  same  way  Sachs  and  Bauer 
say  that  guinea-pig  corpuscles  that  have  been  treated  with  fresh 
horse  serum,  then  washed  and  mixed  with  heated  bovine  serum,  do 
not  deprive  this  latter  of  its  activity.  We  have  repeated  this  exper- 
iment with  different  results;  to  be  sure,  we  have  taken  pains  to  use  a 


450  STUDIES   IN  IMMUNITY. 

much  larger  quantity  of  alexinized  guinea-pig  corpuscles  than  they 
in  order  to  exhaust  the  bovine  serum.  And  we  find  that  bovine 
serum  treated  in  this  manner  becomes,  if  not  absolutely  inactive, 
much  less  active  than  before  treatment. 

In  interpreting  such  experiments  it  is  well  to  note  that  heated 
bovine  serum  acts  in  a  very  small  dose.  A  trace  of  serum,  for  ex- 
ample, 0.01  of  a  cubic  centimeter,  on  addition  to  a  mixture  of  O.o 
of  a  cubic  centimeter  of  salt  solution,  0.3  of  a  cubic  centimeter  of 
fresh  horse  serum  and  0.05  of  a  cubic  centimeter  of  guinea-pig  corpus- 
cles, produces  distinct  conglutination  followed  by  a  certain  amount 
of  hemolysis.  When  we  consider  that  bovine  serum  works  in  such 
small  doses  it  is  easy  to  conceive  how  difficult  it  is  to  deprive  it 
entirely  of  its  active  substance,  the  conglutinin.  To  obtain  such 
distinct  results  one  should  use  much  more  blood  than  do  Sachs  and 
Bauer.  It  is  also  necessary,  particularly  in  the  experiments  with 
bovine  corpuscles,  that  the  blood  should  be  sensitized  and  alexin- 
ized sufficiently.* 

The  details  of  such  an  experiment  with  guinea-pig  corpuscles 
follows : 

In  each  of  two  tubes,  A  and  B,  is  placed  1  c.c.  of  washed  guinea- 
pig  blood.  To  tube  A  is  added  20  c.c.  of  salt  solution  and  6  c.c.  of 
horse  serum.  An  hour  later  the  corpuscles  are  washed,  twice  cen- 
trifugalized  and  the  supernatant  fluids  decanted;  2  c.c.  of  salt 
solution  plus  0.3  of  a  cubic  centimeter  of  heated  bovine  serum  is 
added  to  each  sediment.  An  hour  later  the  mixtures  are  centri- 
fugalized  and  the  supernatant  fluids  A  and  B  decanted.  Another 
mixture  (C)  is  prepared  containing  2  c.c.  of  salt  solution  plus  0.3 
of  a  cubic  centimeter  of  bovine  serum,  56  degrees. 

In  three  tubes,  "a,"  "b,"  "c,"  are  placed  1  c.c.  of  each  fluid,  A, 
B,  and  C  respectively.  To  each  tube  there  is  then  added  0.3  of  a 
cubic  centimeter  of  fresh  horse  serum  and  0.05  of  a  cubic  centi- 
meter of  washed  guinea-pig  blood,  and  the  tubes  are  placed  in  the 
incubator.  The  result  is,  that  in  8  or  10  minutes  "c"  and  "b" 
show  strong  agglutination;  there  is  scarcely  visible  agglutination  in 
"a"  in  20  minutes.  In  a  half  hour,  agglutination,  very  slight,  and 
hemolysis,  scarcely  visible  in  "a,"  strong  agglutination  and  hemoly- 

*  This  is  proved  by  the  fact  that  such  corpuscles  show  no  agglutination  and 
hemolysis  in  the  mixture  of  heated  bovine  serum  and  horse  alexin  unless  they  are 
strongly  sensitized. 


THE  PHENOMENA  OF  ADSORPTION.  451 

sis  in  "b,"  and  strong  agglutination  and  almost  total  hemolysis 
in  "c." 

(/)  Sachs  and  Bauer  take  great  pains  to  interpret,  in  harmony 
with  their  own  ideas,  Bordet  and  Gay's  experiment,  which  shows 
that  bovine  corpuscles,  provided  they  be  sensitized,  are  conglutina- 
ted  and  hemolyzed  by  a  mixture  of  fresh  horse  serum  and  heated 
bovine  serum,  that  is  to  say,  that  they  act  just  as  guinea-pig  corpus- 
cles do.  They  conceive  of  horse  serum  as  containing  several  comple- 
ments, certain  ones  of  which  are  neutralized  by  the  bovine  serum, 
whereas  certain  others  are  not;  as  we  know,  the  Ehrlich  school  has 
no  hesitation  in  multiplying  the  active  substances  in  serum.  We 
believe  it  quite  useless  to  follow  Sachs  and  Bauer  in  their  hypotheses 
concerning  the  peculiarities  of  horse  serum  and  the  reason  that  in 
the  experiment  in  question  the  horse  serum  may  be  replaced  by  any 
other  alexin,  for  example,  by  bovine  alexin  itself,  without  any  effect 
on  the  result.*  As  we  have  already  noted,  when  sensitized  bovine 
corpuscles  are  added  to  fresh  bovine  serum,  the  hemolysis  is 
preceded  by  an  intense  and  characteristic  conglutination.  The 
evolution  of  the  reaction  is  absolutely  the  same  in  both  instances, 
and  it  is  therefore  clear  that  the  mixture  of  fresh  horse  serum, 
(alexin)  and  bovine  serum,  56  degrees  (conglutinin),  acts  exactly 
the  same  as  does  fresh  bovine  serum  (conglutinin  plus  alexin). 
Certain  of  Sachs  and  Bauer's  experiments,  moreover,  in  this  part 
of  their  article  contain  important  experimental  errors. f 

*  Bordet  and  Gay  have  also  described  certain  experiments  in  which  guinea-pig 
alexin  is  employed.  For  example  sensitized  guinea-pig  corpuscles  are  only  slowly 
hemolyzed  by  a  small  dose  of  fresh  guinea-pig  serum.  If  a  little  bovine  serum  is 
added  to  this  mixture,  however,  the  hemolysfs  is  markedly  accelerated  and 
energetic  conglutination  takes  place. 

f  For  example,  in  table  12  of  their  memoir,  Sachs  and  Bauer  say  that  heated 
bovine  serum  is  anticomplementary  for  horse  alexin,  since  it  prevents  the  fixation 
of  this  alexin  on  faintly  sensitized  bovine  corpuscles.  We  shall  not  here  consider 
whether  this  conclusion  is  true  or  false.  What  is  certain  is  that  the  deduction 
from  the  experiment  is  certainly  incorrect.  In  judging  the  intensity  of  alexin 
absorption  Sachs  and  Bauer  place  their  corpuscles,  on  the  one  hand,  in  a  mixture 
of  horse  alexin  and  salt  solution,  and, on  the  other, in  a  mixture  of  horse  alexin  with 
a  certain  amount  of  bovine  serum.  Sachs  and  Bauer  seem  to  forget  that,  other 
things  being  equal,  alexin  fixation,  particularly  when  the  corpuscles  are  faintly 
sensitized,  takes  place  better  in  a  fluid  containing  much  salt  solution  than  in  one 
containing  heated  serum;  this  serum,  as  we  know,  inhibits  alexin  fixation 
without  acting  in  any  truly  anticomplementary  manner.  In  the  question  in  case 
the  heated  bovine  serum  may  inhibit  the  fixation,  not  only  of  horse  alexin,  but 


452  STUDIES   IN  IMMUNITY. 

Beginning  with  their  theory  that  the  bovine  sensitizer  fails  to 
unite  with  guinea-pig  corpuscles  unless  horse  alexin  is  present, 
Sachs  and  Bauer  presuppose  that  a  mixture  of  heated  bovine  serum 
and  horse  alexin  that  has  been  prepared  for  some  time,  and  in  which 
this  combination  may  have  taken  place,  should  hemolyze  guinea- 
pig  corpuscles  more  rapidly  than  a  recently  prepared  mixture. 
They  declare  that  this  can  be  experimentally  proved.  We  have 
repeated  their  attempts  with  different  results.  We  have  noted, 
as  we  shall  later  consider,  that  a  mixture  of  fresh  horse  serum 
and  heated  bovine  serum  in  suitable  proportions  is  much  less 
active  when  it  has  been  kept  for  some  time  before  the  guinea-pig 
corpuscles  are  added. 

Having  examined  Sachs  and  Bauer's  objection,  to  close  the  debate 
we  may  relate  certain  experiments  which  prove  in  an  irrefutable 
manner  the  accuracy  of  Bordet  and  Gay's  interpretation.  The  reason 
that  Ehrlich  and  Sachs'  original  experiment  has  given  rise  to  so  long 
a  discussion  is  due  to  the  multiplicity  of  the  substances  present  and 
particularly  to  the  fact  that  there  are  two  amboceptors  which  can 
supplant  one  another.  The  obscurity  is  due  to  the  fact  that  fresh 
horse  serum  contains  a  sensitizer  as  well  as  does  the  heated  bovine 
serum.  Let  us  suppose,  for  a  moment,  that  horse  serum  contains 
alexin  only.  Under  these  conditions,  on  mixing  the  heated  bovine 
serum  (which,  according  to  Bordet  and  Gay,  is  sensitizing  and,  in 
addition,  contains  the  conglutinin)  with  horse  serum  an  active 
mixture  is  obtained.  We  both  agree  on  this  point.  But  if  we  were 
to  mix  with  such  a  horse  serum  containing  alexin  but  no  sensitizer. 
bovine  serum,  56  degrees r  which  had  been  previously  subjected  to 
guinea-pig  corpuscles,  what  should  we  obtain?  Evidently,  if  Bordet 
and  Gay's  theory  is  correct,  the  mixture  should  be  inactive  because, 
although  such  a  mixture  contains  conglutinin,  it  should  have  been 
deprived  of  all  amboceptor.  If  Ehrlich  and  Sachs  are  correct,  on 
the  contrary,  the  mixture  should  be  just  as  active,  inasmuch  as  it 
should  still,  according  to  these  authors,  contain  the  bovine  sensitizer 
which  can  unite  and  produce  hemolysis  only  in  presence  of  alexin. 

It  would  therefore  be  of  service  to  have  horse  serum  which  con- 
tains alexin  but  is  unable  to  sensitize  guinea-pig  corpuscles.  Such 

also  of  bovine  alexin.  We  shall  later  refer  to  an  experiment  that  proves  this 
fact.  In  addition  it  may  be  mentioned  that  these  facts  have  recently  been  con- 
sidered in  detail  by  Bordet  and  Gay  (see  article,  p.  398). 


THE  PHENOMENA  OF  ADSORPTION.  453 

a  serum  is  easily  obtained,  and  the  experiments  which  have  been 
outlined  confirm  Bordet  and  Gay's  opinion  entirely. 

We  know  that  the  fixation  of  alexin  on  corpuscles  is  very  markedly 
favored  by  the  addition  of  normal  salt  solution;  this  fact  is  very 
evident  in  the  case  of  horse  serum.  Klein  was  first  to  note  that 
guinea-pig  corpuscles  mixed  with  fresh  and  undiluted  horse  serum 
fix  little  alexin,  although  they  absorb  it  completely  when  a  con- 
siderable amount  of  salt  solution  is  added.  The  sensitizer,  on 
the  contrary,  is  well  absorbed  even  when  the  serum  is  concentrated. 
Consequently,  if  we  treat  undiluted  horse  serum  with  an  equal 
volume  of  washed  guinea-pig  corpuscles,  we  shall  obtain  a  fluid 
which  is  rich  in  alexin  but  is  deprived  of  sensitizer. 

We  find  that  such  a  serum  produces  the  following  effect: 
(a)     When  mixed  with  intact  heated  bovine  serum  and  guinea- 
pig  corpuscles  it  produces  the  phenomenon  of  conglutination  and 
hemolysis  very  clearly.     This  fact  proves  that  the  bovine  sensi- 
tizer is  the  one  which  acts,  as  no  other  one  is  present. 

(6)  When  mixed  with  heated  bovine  serum  that  has  been  pre- 
viously treated  with  guinea-pig  corpuscles  it  produces  no  phe- 
nomenon. This  proves  that  such  a  mixture  contains  no  sensitizer 
at  all,  and  consequently  condemns  Ehrlich  and  Sachs'  thesis,  in 
accordance  with  which  the  bovine  sensitizer  should  still  be  present. 
In  control  tubes,  of  course,  it  is  proved  that  such  heated  bovine 
serum,  when  mixed  with  untreated  horse  serum,  gives  the  phe- 
nomenon perfectly,  in  which  case,  of  course,  the  sensitizer  that 
acts  is  in  the  horse  serum. 

(c)  When  mixed  with  heated  bovine  serum  that  has  been 
deprived  of  its  amboceptor  by  contact  with  guinea-pig  corpuscles 
this  horse  serum  deprived  of  its  sensitizer  still  gives  the  phenomenon 
if  the  corpuscles  added  are  not  normal,  but  have  first  been  sensi- 
tized by  contact  with  heated  bovine  serum  and  subsequently 
washed.  This  completes  the  demonstration  of  the  previous  experi- 
ment on  the  fixation  of  the  bovine  sensitizer  by  corpuscles  in 
absence  of  alexin.  The  experimental  details  of  these  experiments 
follow.  Materials  are  prepared  as  follows: 

I.  Fresh  horse  serum  containing  alexin  but  deprived  of  sensi- 
tizer. —  2  c.c.  of  washed  guinea-pig  blood  is  added  to  2  c.c.  of  horse 
serum.  After  contact  for  quarter  of  an  hour  the  mixture  is  cen- 


454 


STUDIES   IN  IMMUNITY. 


trifugalized,  the  supernatant  fluid  decanted,  and  G  c.c.  of  normal 
salt  solution  added. 

II.  Fresh  horse  serum  deprived  of  both  sensitizer  and  alexin.  — 
6  c.c.  of  salt  solution  plus  2  c.c.  of  washed  guinea-pig  blood  is  added 
to  2  c.c.  of  the  serum.     After  contact  for  an  hour  and  a  half  the 
mixture  is  centrifugalized  and  the  supernatant  fluid  decanted. 

III.  Bovine  serum  (56  degrees)  deprived  of  its  sensitizer.  — 2  c.c. 
of  salt  solution  plus  2  c.c.  of  washed  guinea-pig  blood  is  added  to 
2  c.c.  of  the  serum.     Contact  for  1  hour,  centrifugalization  and 
decantation  of  the  supernatant  fluid. 

IV.  Intact  bovine  serum,  56  degrees. — 2  c.c.  of  serum  plus  4 
c.c.  of  salt  solution. 

V.  Intact  horse  serum. — 2  c.c.  of  serum  plus  8  c.c.  of  salt  solu- 
tion. 

Mixtures  are  prepared  as  indicated  in  the  following  table  from 
these  fluids,  and  0.05  of  a  cubic  centimeter  of  normal  guinea-pig 
corpuscles,  or  of  corpuscles  that  have  previously  been  treated  with 
0.3  of  a  cubic  centimeter  of  heated  bovine  serum  and  subse- 
quently carefully  washed  three  times  with  a  large  volume  of  salt 
solution,  is  then  added. 

Mixtures  in  which  the  characteristic  intense  agglutination  and 
hemolysis  occurred  are  designated  by  the  letters  AH.  In  the 
other  mixtures  the  corpuscles  have  shown  no  such  modifications 
even  after  a  considerable  period  of  time. 

TABLE  I. 


0.6  c.c.  of  fluid  con- 
taining bovine  serum. 

Normal  guinea-pig  corpuscles, 
0.05  c.c.;  1.5  c.c.  of  a  fluid  con- 
taining horse  serum. 

0.05  c.c.  of  corpuscles  sensitized 
by  bovine  serum  and  washed. 
Fluids  contain  1.5  of  horse  serum. 

I. 
Minus 
sensitizer. 

II. 

Minus 
sensitizer 
and  iilfxiii. 

V. 

Intact. 

I. 

Minus 
sensitizer. 

II. 

Minus 
sensitizer 
and  alexin. 

Intact. 

III.  Bovine  serum 
minus  sensitizer 

No.  1 

0 

No.  2 
0 

No.  3 
AH 

No.  10 
AH 

No.  11 

0 

No.  12 
AH 

IV.  Intact  bovine 
serum 

No.  4 
AH 

No.  5 
0 

No.  6 
AH 

No.  13 
AH 

No.  14 
0 

No.  15 
AH 

0.6  c.c.  salt  solu- 
tion   

No.  7 
0 

No.  8 
0 

No.  9 

0 

No.  16 

0 

No.  17 

0 

No.  18 
0 

THE  PHENOMENA  OF  ADSORPTION.  455 

We  may  add  that  the  previous  sensitization  of  the  corpuscles 
is  of  considerable  importance  in  respect  to  the  rapidity  with  which 
the  conglutination  and  hemolysis  appear.  Thus  we  find  that  in- 
tense agglutination  takes  place  in  from  5  to  7  minutes  in  tubes 
12,  13  and  15,  in  10  minutes  in  tube  10  and  in  about  15  minutes 
in  tubes  3,  4  and  6.  The  rapidity  of  hemolysis  corresponds. 

The  contrast  between  tubes  1  and  10,  1  and  4,  and  1  and  3  is 
particularly  to  be  noted. 

Similar  experiments  made  with  fresh  bovine  serum  give  similar 
results.  Inasmuch  as  alexin  fixation  follows  the  fixation  of  the 
sensitizer,  by  placing  fresh  bovine  serum  in  contact  with  an 
equal  volume  of  washed  guinea-pig  blood  for  a  short  period 
(about  10  minutes  at  room  temperature)  we  obtain  a  fluid  that 
is  deprived  of  sensitizer  but  still  contains  an  appreciable  amount 
of  alexin  and  of  conglutinin.  Serum  treated  in  this  manner  has 
no  effect  on  normal  guinea-pig  corpuscles,  but  it  does  hemolyze  cor- 
puscles that  have  been  previously  sensitized  by  heated  bovine  serum 
and  subsequently  washed,  and  the  hemolysis  in  this  case  also  is 
preceded  by  the  characteristic  conglutination.  In  the  same  way 
we  find  that  a  relatively  small  dose  of  fresh  intact  bovine  serum, 
when  added  to  guinea-pig  corpuscles,  conglutinates  and  hemolyzes 
them  more  rapidly  if  they  have  been  previously  sensitized  and 
washed.  For  example,  in  two  tubes,  A  and  B,  is  placed  0.1  of  a 
cubic  centimeter  of  washed  guinea-pig  blood  and  0.5  of  a  cubic 
centimeter  of  salt  solution.  To  A  is  then  added  0.5  of  a  cubic 
centimeter  of  heated  bovine  serum.  A  half  hour  later  the  cor- 
puscles are  washed,  centrifugalized  and  the  supernatant  fluid  de- 
canted; 1  c.c.  of  salt  solution  and  0.1  of  a  cubic  centimeter  of 
fresh  bovine  serum  is  then  added  to  each  corpuscle  sediment.  In 
tube  A  conglutination  takes  place  in  40  minutes  and  hemolysis  in 
an  hour;  in  B  the  phenomena  occur  in  1  and  2  hours  respectively. 
It  is  certain,  then,  that  guinea-pig  corpuscles  that  have  been  sub- 
jected to  bovine  serum,  56  degrees,  fix  the  sensitizer. 

We  may  now  recall  in  this  connection  the  fact  which  we  have 
already  mentioned  and  which  at  first  sight  might  seem  contra- 
dictory to  the  preceding  statements.  If  we  prepare  a  mixture 
containing  a  large  amount  of  salt  solution  with  a  little  fresh  in- 
tact bovine  serum,  the  addition  of  sensitizer  in  the  form  of  heated 


456  STUDIES  IN  IMMUNITY. 

bovine  serum  may  inhibit  the  hemolysis  of  guinea-pig  corpuscles 
rather  than  favor  it.*  We  found  indeed  that  alexin  fixation  of  the 
sensitized  corpuscles  takes  place  better  when  the  fluid  contains  little 
serum  and  a  large  amount  of  salt  solution.  For  example,  we  pre- 
pare two  tubes,  A  and  B,  containing  each  0.8  of  a  cubic  centimeter 
of  salt  solution  plus  0.1  of  a  cubic  centimeter  of  fresh  bovine  serum. 
To  A  we  add  0.3  of  a  cubic  centimeter  of  bovine  serum,  56  degrees,  and 
to  both  tubes  0.05  of  a  cubic  centimeter  of  washed  guinea-pig  blood. 
Conglutination  and  hemolysis  occur  in  tube  B  in  30  and  40  minutes 
respectively,  in  tube  A  in  40  and  60  minutes.  In  other  words,  the 
heated  serum  is  more  inhibiting  as  serum  than  it  is  useful  as  sen- 
sitizer,  inasmuch  as  enough  sensitizer  is  present  in  the  fresh  bovine 
serum.  But  if,  instead  of  employing  in  this  last  experiment  fresh 
intact  bovine  serum,  we  use  serum  that  has  been  treated  for  about 
10  minutes  with  an  equal  volume  of  guinea-pig  blood,  we  obtain 
directly  opposite  results.  Under  such  conditions  the  addition  of 
a  sensitizer  is  obviously  necessary;  and  consequently  we  find  that 
although  the  heated  bovine  serum  (0.3  of  a  cubic  centimeter),  as 
serum,  inhibits  alexin  fixation,  it  is  necessary  as  containing  sensi- 
tizer. An  experiment  modified  in  this  manner  shows  in  tube  B 
no  conglutination  or  hemolysis;  the  phenomena  appear  in  A  in 
1J  and  2  hours  respectively.  It  would  seem  to  us  in  view  of  so 
many  convincing  facts  that  the  thesis  of  Ehrlich  and  Sachs,  which 
supposes  that  the  bovine  amboceptor  can  unite  with  the  cor- 
puscles only  in  presence  of  the  alexin,  can  no  longer  be  admitted. 
And  what  is  more,  we  consider  these  authors'  opinions  on  hemoly- 
sis as  purely  theoretical,  in  so  far  as  they  bear  on  the  existence 
of  a  complementophilic  group,  partial  anticomplements,  multi- 
plicity of  complements,  dominant  and  non-dominant  complements, 
and  the  like. 

SECTION  II. 

VARIOUS  EXAMPLES  OF  CONGLUTINATION  AND  THE  MODE 
OF  ACTION  OF  THE  CONGLUTININ. 

Conglutination  always  appears  when  the  cells  added  to  fresh 
bovine  serum  or  a  mixture  of  heated  bovine  serum  and  alexin  from 
another  animal  are  sufficiently  sensitized  and  consequently  in  a 
condition  to  absorb  alexin.  Whenever  the  normal  sera  employed 

*  See  the  previous  footnote  criticising  certain  experiments  of  Sachs  and  Bauer. 


THE  PHENOMENA  OF  ADSORPTION.  457 

in  this  mixture  are  not  sensitizing  for  the  corpuscles  used,  they 
must  be  treated  with  an  appropriate  specific  immune  serum.  Such 
is  the  case  in  the  experiment  with  bovine  corpuscles.  Many 
corpuscles,  however,  react  sufficiently  to  the  sensitizer  in  normal 
serum,  particularly  in  bovine  serum.  Thus  in  Ehrlich  and  Sachs' 
experiment  the  guinea-pig  corpuscles  may  be  replaced  by  other 
red  blood  cells.  For  example,  rabbit  corpuscles  act  just  the  same 
as  guinea-pig  corpuscles  and  show  conglutination,  although  some- 
what less  rapidly,  and  intense  hemolysis.  Human  corpuscles 
react  distinctly.  In  all  these  instances  fresh  bovine  serum  alone 
produces  similar  results. 

Goat  corpuscles  act  rather  curiously  on  the  addition  of  fresh 
bovine  serum.  They  are  energetically  conglutinated,  but  are  not 
hemolyzed.  We  should  conclude  from  this  fact  that  the  bovine 
serum  sensitizes  goat  corpuscles  sufficiently  to  produce  alexin 
absorption  and  bring  about  the  action  of  the  conglutinin,  but  that 
the  alexin  acts  poorly  in  producing  hemolysis,  owing  perhaps  to  the 
fact  that  the  ox  and  the  goat  are  closely  related  species.*  In  the 
phenomena  under  consideration,  hemolysis  is  due  neither  to  the 
alexin  nor  to  the  conglutinin  alone,  but  to  the  combined  effect  of 
the  two  factors;  the  complex  alexin-conglutinin  does  the  hemoly- 
zing,  and  we  shall  see  presently  that  the  conditions  under  which 
this  complex  is  formed  are  of  importance  in  respect  to  the  strength 
of  the  hemolytic  or  the  agglutinating  property  which  it  produces. 

Why  does  the  sensitized  and  alexinized  corpuscle  unite  with 
the  conglutinin?  The  absorption  of  alexin  (complement)  by  a 
sensitized  corpuscle  is  due,  as  we  know,  to  a  phenomenon  of  ad- 
sorption. But  to  what  is  this  adsorption  due?  Neither  the 
corpuscle  itself  nor  the  sensitizer  alone  shows  any  such  attrac- 
tion for  the  alexin.  The  corpuscle  and  the  sensitizer  when  they 
unite,  however,  form  a  complex  which  is  endowed  with  the  new 
property  of  alexin  absorption,  owing  doubtless  to  the  fact  that 
the  sensitizer  produces  a  change  of  physical  condition  in  the  cor- 
puscle and  sort  of  coagulation  similar  to  that  brought  about  by 
adding  specific  precipitating  serum  to  an  albuminous  solution. 
This  formation  of  a  precipitate  would  affect  the  contact  proper- 

*  Bovine  alexin  fails  to  hemolyze,  or  hemo-lyzes  only  faintly,  goat  corpuscles 
even  when  they  are  sensitized  by  a  specific  immune  serum. 


458  STUDIES  IN  IMMUNITY. 

ties  markedly,  and  in  particular  would  produce  an  avidity  for 
alexin  absorption.  Thanks  to  the  researches  of  Gengou  and  of 
Gay,  we  know  that  the  albuminous  substances  of  serum,  of  milk 
and  the  like,  become  avid  of  alexin  when  precipitins  are  added. 

It  does  not  seem,  a  priori,  necessary  that  in  each  instance  the 
same  result  should  occur.  In  certain  other  cases  the  facility  of 
different  substances  to  clump  together  may  be  supposed  to  be  due 
to  the  presence  of  such  tendencies  of  adhesion  in  each  separate 
substance.  For  example,  if  A,  B  and  C  clump  one  another  it 
may  be  supposed  that  one  of  these  elements,  B  for  example, 
shows  a  property  of  adhesion  both  toward  A  and  toward  C,  and 
may  show  an  intermediary  function  in  producing  the  complex.* 

It  would  seem,  in  the  case  of  conglutination,  that  alexin  fills 
such  an  intermediary  function.  It  seems  to  unite  on  the  one  hand 
with  the  sensitized  corpuscle,  as  we  already  know,  but  it  is  also 
evident  that  there  is  some  reaction  between  it  and  the  conglutinin. 
To  demonstrate  this  fact,  we  may  take  fresh  horse  serum,  pre- 
viously diluted  with  an  equal  volume  of  salt  solution,  and  heated 
bovine  serum  diluted  in  the  same  manner.  We  add  1  c.c.  of  the 
diluted  bovine  serum  to  say  0.2  of  a  cubic  centimeter  of  diluted 
horse  serum.  This  mixture  is  left  for  a  time  at  room  tempera- 
ture. After  a  while  another  similar  mixture  is  prepared,  and  0.05 
of  a  cubic  centimeter  of  washed  guinea-pig  corpuscles  is  then 
added  to  each  of  them.  We  find  that  the  first  mixture,  in  which 
a  longer  contact  has  taken  place,  has  lost  its  property  of  producing 
conglutination  and  hemolysis  almost  entirely;  in  the  second  mix- 
ture the  phenomena  occur  well. 

The  experiment  'may  be  repeated  with  modifications  in  the  re- 
spective doses  of  each  serum  or  in  the  duration  of  contact.  In  the 
doses  we  have  given,  a  rather  short  contact  suffices  to  diminish 
the  activity  of  the  mixture,  even  10  or  15  minutes  being  sufficient 
to  produce  a  distinct  difference.  As  regards  the  dosage,  it  is  found 
that  the  depressing  effect  of  contact  becomes  less  and  less  distinct 
as  the  quantity  of  the  alexin  is  increased  relative  to  the  amount 
of  heated  bovine  serum.  For  example,  in  a  mixture  of  equal  parts 

*  If  this  were  so,  B  might  well  deserve  the  name  of  amboceptor,  which  Ehrlich 
has  incorrectly  applied  to  sensitizers,  with  the  reservation,  however,  that  we  are 
now  dealing  with  tendencies  to  adhesion  and  not  with  purely  chemical  affinities. 


THE  PHENOMENA  OF  ADSORPTION.  459 

of  the  two  sera  diluted  each  with  an  equal  amount  of  salt  solution, 
the  corpuscles  react  very  much  as  they  do  in  a  fresh  mixture. 

Ehrlich's  theories  would  feel  no  embarrassment  in  face  of  such 
a  fact.  To  explain  it,  we  may  suppose  that  bovine  serum  con- 
tains several  amboceptors,  among  which  are  some  with  no  affinity 
for  the  guinea-pig  corpuscles.  Such  amboceptors,  if  time  were 
given  them,  would  monopolize  the  alexin,  would  deviate  it,  and 
prevent  it  from  uniting  with  the  guinea-pig  corpuscle.  If  the 
experimental  result  had  turned  out  in  the  opposite  way,  the  same 
theories  would  still  be  satisfied.  It  would  suffice  to  say  simply 
that  the  bovine  amboceptor  which  combines  with  the  guinea-pig 
corpuscles  shows  a  greater  affinity  when  it  has  had  time  to  unite 
previously  with  alexin.  As  we  have  already  seen,  Sachs  and  Bauer 
have  suggested  this  interpretation  in  similar  experiments  which 
prove  to  them  in  some  inexplicable  manner  that  a  prolonged  con- 
tact exaggerates  the  activity  of  a  mixture  instead  of  diminishing 
it.*  As  long  as  one  has  recourse  to  such  purely  hypothetical  ideas, 
which  may  be  varied  and  multiplied  as  one  desires,  it  is  clear  that 
any  experimental  fact  may  be  interpreted.  The  statement  that 
the  amboceptor  has  a  complementophilic  group  is  a  hypothesis. 
The  statement  that  the  affinity  of  this  group  varies  with  different 
amboceptors  is  another  hypothesis;  the  supposition  that  the  cyto- 
philic  group  becomes  chemically  more  active  when  affinities  of 
the  complementophilic  group  are  satisfied  is  a  third  hypothesis. 
A  theory  is  of  service  only  when  used  to  coordinate  real  and  demon- 
strated facts;  it  is  of  no  value  when  it  unites  hypothetical  facts 
produced,  created  and  fashioned  at  the  will  of  the  theorist,  with 
a  single  demonstrated  fact. 

The  question  of  the  mutual  relations  between  the  sensitized 
corpuscle,  the  alexin  and  the  conglutinin  should  be  subjected  to 
additional  researches  in  order  to  be  quite  clear.  It  seems,  how- 
ever, that  to  obtain  the  maximal  effect  of  agglutination,  and  par- 
ticularly of  hemolysis,  the  order  in  which  the  three  substances 
are  added  should  be  kept  in  mind.  If  the  conglutinin  and  alexin 
are  added  in  certain  proportions  and  an  interval  allowed  to  elapse 

*  Sachs  and  Bauer's  mixtures,  to  be  sure,  contained  relatively  more  horse 
serum  than  ours.  We  have  not,  however,  been  able  to  produce  their  results  even 
by  following  their  experiment  closely. 


460  STUDIES  IN   IMMUNITY. 

before  the  corpuscles  are  added,  only  slight  effects  are  produced. 
If  the  fixation  of  the  alexin  on  the  corpuscles  is  brought  about, 
and  they  are  then  washed  and  subsequently  subjected  to  the  con- 
glutinin,  they  are  clumped  but  not  hemolyzed.*  The  effects  vary 
markedly,  according  to  the  modus  operandi,  although  the  sub- 
stances concerned  remain  the  same.  This  is  not  surprising,  for 
we  find  similar  examples  throughout  the  phenomena  of  molecular 
adhesion.  Toxin  and  antitoxin  when  mixed  in  constant  pro- 
portions can,  as  we  know,  furnish  complexes  endowed  with  differ- 
ent properties  depending  on  whether  they  are  added  to  one  another 
all  at  once,  or  in  divided  doses.  In  the  serum  diagnosis  of  syphilis, 
for  example,  the  slightest  details  in  the  preparation  of  the  liver 
extract  is  of  importance.  Sachs  and  Rondoni  have  found,  for 
example,  that  their  results  vary  in  accordance  with  whether  their 
concentrated  alcoholic  extract  of  liver  is  diluted  with  salt  solu- 
tion rapidly  or  slowly.  It  is  conceivable,  as  these  authors  note, 
that  the  active  substance  may  be  carried  to  different  states  of 
colloidal  division  in  accordance  with  these  varying  conditions, 
and  that  differences  of  a  physical  nature  correspond  to  the  various 
results  in  the  energy  of  alexin  adsorption. 

The  physical  condition  of  the  conglutinin  would  seem,  likewise, 
to  have  a  distinct  influence  on  the  result  of  the  phenomenon.  On 
dialyzing  bovine  serum,  56  degrees,  we  obtain  by  centrifugalization 
a  supernatant  fluid  and  a  precipitate,  the  properties  of  which  vary, 
although  they  both  contain  conglutinin.  On  adding  sufficient 
sodium  chlorid  to  reestablish  the  primitive  tonicity,  we  find  that 
the  supernatant  fluid,  when  added  to  fresh  horse  serum,  hemolyzes 
guinea-pig  corpuscles  better  than  it  conglutinates  them.f  The 
precipitate  gives  the  opposite  result.  When  washed  in  distilled 
water  and  shaken  in  salt  solution,  this  precipitate  dissolves  only 
partially;  a  cloudy  fluid  is  obtained,  which,  on  the  addition  of  fresh 
horse  serum,  agglutinates  guinea-pig  corpuscles  energetically,  but 
hemolyzes  them  poorly.  A  mixture  of  the  supernatant  fluid  and 

*  Several  examples  of  this  fact  have  been  noted  in  the  articles  of  Bordet  and 
Gay  and  of  Sachs  and  Bauer. 

t  In  certain  cases,  however,  this  fluid  may  give  agglutination  without  hemolysis 
as  does  heated  bovine  serum.  This  occurs  when  such  a  fluid  is  added  to  guinea- 
pig  corpuscles  that  have  been  previously  subjected  to  fresh  horse  serum  and 
subsequently  washed. 


THE  PHENOMENA  OF  ADSORPTION.  461 

the  precipitate  reproduces  a  mixture  which  is  very  like  normal 
bovine  serum. 

In  view  of  this  relative  dissociation  of  agglutination  and  hemoly- 
sis,  it  may  be  that  the  conglutinin  is  not  a  simple  substance,  or 
that  it  is  a  simple  substance  occurring  in  different  physical 
conditions  or  conditions  of  greater  or  less  solution  in  serum.  If 
we  like,  we  may  regard  it  as  a  more  or  less  fine  colloid  solution. 
This  fact  is  evidently  important  to  study.  The  fact  of  interest 
for  the  moment  is,  as  we  have  brought  out  again  in  this  article,  that 
the  hemolysis  and  conglutination  by  bovine  serum  follow  a  general 
law. 

To  summarize:  The  conclusions  of  the  present  article  corrob- 
orate the  ideas  of  Bordet  and  Gay  of  two  years  ago.  The  objec- 
tions that  have  been  offered  to  these  ideas  by  Sachs  and  Bauer 
have  no  foundation.  Contrary  to  the  opinions  of  Ehiiich  and 
Sachs,  bovine  sensitizers  unite  perfectly  well  with  corpuscles  when 
the  alexin  is  absent.  The  hypothesis  of  the  existence  of  a  com- 
plementophilic  group  in  sensitizers  does  not  agree  with  the  facts. 
As  for  the  special  properties  of  bovine  serum,  they  are  due  to  a 
particular  substance,  the  conglutinin,  the  principal  characters 
of  which  are  now  clearly  brought  out.  We  may  add  that  this 
substance  is  precipitated  for  the  greater  part  by  dialysis  and  seems 
to  manifest  a  real  affinity  for  alexin,  and  therefore  tends  to  pre- 
cipitate itself  on  sensitized  and  alexinized  corpuscles. 


XXIV.    SENSITIZERS  FOR  THE  TUBERCLE  BACILLUS.* 

BY   J.   BORDET  AND   O.   GENGOU. 

One  of  us  demonstrated  in  1900  that  if  red  blood  cells  or  bacteria 
are  added  to  a  specific  immune  serum  which  contains  a  specific  sen- 
sitizer, the  cells  become  capable  of  absorbing  the  destructive  sub- 
stances in  the  serum  (alexin).  With  this  fact  as  a  basis,  Bordet 
^and  Gengou  have  described  a  method  which  allows  one  to  prove 
the  existence  of  a  sensitizer  in  a  given  serum.  By  preparing  a 
suitable  mixture  of  typhoid  bacilli,  fresh  normal  human  serum, 
and  heated  serum  (55  degrees)  of  a  convalescent  from  typhoid,  it 
is  found  that  the  alexin  of  the  normal  serum  is  absorbed  by  the 
bacilli;  this  is  proved  by  the  fact  that  subsequently  added  sen- 
sitized corpuscles  undergo  no  hemolysis.  Consequently  we  may 
conclude  that  the  serum  of  patients  convalescent  from  typhoid  con- 
tains a  sensitizer  that  endows  the  typhoid  bacillus  with  the  power 
of  fixing  alexin.  By  this  method  we  have  endeavored  to  determine 
whether  a  guinea-pig  can  form  an  active  sensitizer  for  the  tubercle 
bacillus;  our  results  follow: 

Guinea-pigs  injected  with  living  human  tubercle  bacilli  soon 
show  generalized  tuberculosis  and  produce  no  sensitizer.  A  nega- 
tive result  is  obtained  in  any  stage  of  their  disease.  If  the  guinea- 
pigs  receive  a  subcutaneous  inoculation  of  the  avian  tubercle 
bacillus,  on  the  contrary,!  for  two  or  three  times  they  resist  infec- 
tion, and  soon  form  a  sensitizer  in  their  blood  which  has  the  property 
of  provoking  alexin  fixation  when  added  to  the  bacillus.  It  is 
interesting  to  note  that  this  sensitizer  shows  an  equal  activity 
with  the  human  tubercle  bacillus;  the  same  amount  of  sensitizing 
serum  will  fix  the  same  amount  of  alexin  with  the  same  volume 

*Les  sensibilisatrices  du  bacille  tuberculeux:  Comptes  Rendus  de  I'Acad&nie 
des  Sciences,  vol.  137,  1903,  351. 

t  Our  culture  of  avian  bacillus  was  derived  from  pigeons  and  had  been  grown 
for  some  time  on  glycerinated  potato. 

462 


SENSITIZERS  FOR  THE  TUBERCULE  BACILLUS.  463 

of  either  human  or  avian  bacilli.  In  other  words,  the  serum  ob- 
tained following  an  injection  of  avian  bacilli  does  not  give  a  means 
of  distinguishing  the  two  races  of  tubercle  bacillus. 

If  guinea-pigs  are  given  injections  of  a  mixture  of  human  tubercle 
bacilli,  killed  by  heating  to  70  degrees,  and  sensitizing  serum,  fol- 
lowed at  the  end  of  2  weeks  by  another  similar  mixture  con- 
taining simply  dried  bacilli,  we  find  that  the  animals  have  become 
more  resistant  than  controls  to  the  living  human  tubercle  bacillus. 
Such  treated  animals,  on  receiving  an  injection  of  this  organism, 
survive  considerably  longer  than  do  the  controls,  but  when  killed 
about  3  months  after  injection,  their  organs  are  found  filled 
with  tubercles,  notwithstanding;  in  other  words,  the  rapidity  of 
the  disease  is  simply  checked.  The  serum  of  such  animals  tested 
at  this  period  shows  the  presence"  of  a  sensitizer.  The  sensitizing 
property,  then,  although  not  quite  useless,  is  incapable  of  prevent- 
ing the  evolution  of  the  disease.  Guinea-pigs  that  have  been 
treated  simply  with  injections  of  human  bacilli  killed  by  heating 
to  70  degrees,  followed  by  dried  bacilli,  also  acquire  a  sensitizing 
property  in  their  serum,  but,  as  we  have  already  known  for  some 
time,  their  resistance  to  the  living  bacillus  has  not  been  remark- 
ably increased. 


XXV.      NEW  CONTRIBUTION  TO  THE  STUDY  OF  SEN- 
SITIZERS  FOR  TUBERCLE  BACILLI.* 

BY  DR.   O.   GENGOU. 

Bordet  and  Gengout  showed  that  guinea-pigs  inoculated  with 
virulent  human  tubercle  bacilli  die  without  forming  any  sensi- 
tizers  for  this  bacillus,  but  if,  on  the  contrary,  they  receive  an 
injection  of  living  avian  tubercle  bacilli,  for  which  organisms  they 
are  usually  resistant,  they  form  sensitizers  that  act  not  only  on 
the  avian  bacillus,  but  also  on  the  human  bacillus.  Later  on, 
DembinskiJ  stated  that  the  living  human  bacillus  produces  no 
sensitizer  either  in  the  rabbit  or  in  the  pigeon,  and  he  concludes 
"that  the  production  of  sensitizers  for  tubercle  bacilli  does  not 
depend  on  the  greater  or  less  resistance  of  the  animal  employed 
against  these  bacilli,"  as  Bordet  and  Gengou  believed,  "but  is 
related  to  the  type  of  bacilli  employed." 

In  the  course  of  studies  on  experimental  tuberculosis,  we  have 
had  occasion  to  control  Dembinski's  results.  Since  guinea-pigs 
succumb  to  an  inoculation  of  virulent  human  bacilli  without  pro- 
ducing sensitizers,  we  used  killed  human  bacilli  (heated  for  one- 
half  hour  to  65  degrees  or  for  5  minutes  to  100°  C.).  For  compari- 
son we  gave  other  guinea-pigs  injections  of  avian  bacilli  killed 
in  the  same  manner.  Similar  injections  were  also  made  in  rabbits. 
The  animals  were  all  immunized  on  three  successive  occasions 
by  subcutaneous  injections  at  intervals  of  3  weeks;  they  were  bled 
2  or  3  weeks  after  the  last  injection. 

We  determined  the  presence  of  sensitizers  in  the  serum  of  these 
animals  by  the  Bordet-Gengou  method  based  on  the  characteristic 

*  Nouvelle  contribution  a  lY'tude  des  sensibilisatrices  des  bacilles  tuberculeux. 
Comptes  Rend,  de  la  Soc.  de  Biol.,  LVIII,  1906,  218. 

t  Gengou,  p.  462.     Comptes  Rendus  de  TAcad^mie  des  Sciences. 
J  Dembinski,  Soci^te"  de  Biologic,  1901. 

464 


CONTRIBUTION  TO  THE  STUDY  OF  SENSITIZERS. 


465 


property  of  sensitizers  to  fix  alexin  on  the  cell  for  which  the  sen- 
sitizer  is  specific.  If  a  given  serum  contains  an  active  sensitizer 
for  the  tubercle  bacillus,  the  alexin  will  be  fixed  by  this  bacillus, 
and  subsequently  introduced  sensitized  red  blood  cells  will  remain 
intact,  owing  to  the  absence  of  free  alexin. 

A  resume  of  our  results  is  given  in  the  following  table.  Apart 
from  control  tubes,*  the  composition  of  which  is  too  complicated 
to  write  out  in  detail,  each  experiment  comprises  a  tube  that  con- 
tains 0.1  of  a  cubic  centimeter  of  guinea-pig  alexin,  0.6  of  a  cubic 
centimeter  of  heated  serum  and  0.2  of  a  cubic  centimeter  of  an 
emulsion  of  fresh  human,  avian,  or  bovine  bacilli,  as  the  case  may 
be.  Three  hours  later  0.1  of  a  cubic  centimeter  of  sensitized  goat 
corpuscles  is  added  to  each  tube.f 

TABLE  I. 


1. 

2. 
3. 

4. 
5. 
6. 

7. 
8. 

Serum  of  guinea-pig  injected  with  human  T.  B.  65°.  .  .  . 
Serum  of  guinea-pig  injected  with  human  T.  B.  100°.  .  . 
Serum  of  guinea-pig  injected  with  avian  T.  B.  65°  
Serum  of  guinea-pig  injected  with  avian  T.  B.  100°.  .  .  . 
Serum  of  rabbit  injected  with  human  T.  B.  65°  

Fixation  with  emul- 
sion of  T.  B.  bacilli. 

Human 

Avian 

Bovine 

H-  1  +K-  +  +  +  + 

H-  1  +H-  +  +  +  + 

± 

Serum  of  rabbit  injected  with  human  T.  B.  100°  

Serum  of  rabbit  injected  with  avian  T.  B.  65°  

Serum  of  rabbit  injected  with  avian  T.  B.  100°  

+  =  Complete  fixation;  strong  sensitizer 
±=  Partial  fixation;  weak  sensitizer 
—  =  No  fixation;  no  sensitizer 

As  a  result  of  our  researches,  we  find  that  human  or  avian  bacilli 
killed  by  heating  to  65  degrees  or  100  degrees  stimulate  the  forma- 
tion in  guinea-pigs  of  sensitizers  active  against  the  various  mam- 
malian tubercle  bacilli.  This  stimulation  of  sensitizer  formation 

*  Bordet  and  Gengou,  p.  217. 

t  In  these  experiments  washed  goat  corpuscles  are  sensitized  by  an  equal 
volume  of  rabbit-antigoat  serum,  and  after  contact  for  a  quarter  of  an  hour  again 
washed  in  salt  solution;  the  corpuscles  are  then  suspended  in  a  double  quantity 
of  0.85  per  cent  salt  solution. 


466  STUDIES  IN   IMMUNITY. 

is  also  possible  in  a  rabbit,  particularly  with  human  bacilli  that  have 
been  heated  to  100  degrees,  but,  as  is  evident  from  the  table,  it  is 
more  difficult  to  produce.*  The  facts  that  we  have  observed, 
however,  as  regards  the  rabbit,  and  particularly  in  the  guinea- 
pig,  prevent  our  adherence  to  Dembinski's  opinion,  who  thinks 
in  regard  to  the  rabbit  and  the  pigeon  that  "the  injection  of 
killed  bacilli,  whether  human  or  avian,  produces  no  sensitizer  in 
their  blood." 

We  feel  justified  in  concluding  from  our  results  that,  contrary 
to  Dembinski's  opinion,  tlie  production  of  antituberculous  sensiti- 
zers  does  not  depend  on  the  type  of  bacilli  injected.  The  human 
bacillus  as  well  as  the  avian  bacillus,  and  acid-resisting  bacilli 
in  general,  may  give  rise  on  injection  in  the  rabbit,  and  par- 
ticularly in  the  guinea-pig,  to  sensitizers  that  are  active  for  the 
various  types  of  mammalian  tubercle  bacilli,  as  we  shall  later  show. 
It  may  be,  however,  on  the  other  hand,  that  the  facility  with  which 
these  antibodies  are  formed  depends  on  the  animal  species  em- 
ployed ;  and  the  guinea-pig  would  seem  to  yield  them  more  readily 
than  does  the  rabbit,  or,  according  to  Dembinski,  the  pigeon. 

We  have  been  able  to  confirm  the  other  results  of  this  writer, 
who  found  that  antituberculous  sensitizers  are  just  as  active  when 
heated  human  or  avian  bacilli  (one-half  hour  to  65  degrees)  are 
employed. 

*  The  slight  activity  of  the  serum  from  the  majority  of  our  vaccinated  rabbits 
may  be  due  simply  to  insufficient  immunization;  we  are,  for  the  moment, 
engaged  in  a  study  along  this  line. 


XXVI.    A  CONTRIBUTION  TO  OUR  KNOWLEDGE  OF  ANTI- 
TUBERCULOUS  SENSITIZERS. 

BY  DR.   GENGOU. 

Since  Bordet's  researches  on  the  mechanism  of  acquired  im- 
munity, our  knowledge  of  the  sensitizers  (Ehrlich's  amboceptors) 
has  come  to  be  more  and  more  definite.  These  substances,  which 
appear  in  the  blood  of  inoculated  animals  during  the  course  of 
immunization,  are,  as  we  know,  specific  and  have  as  a  peculiar 
property  the  power  of  fixing  the  alexin  on  that  substance  against 
which  the  animal  that  furnishes  the  sensitizers  has  been  immunized. 
The  alexin  of  normal  serum  alone  has  little  or  no  tendency  to  be- 
come fixed  on  bacteria  or  red  blood  cells ;  a  bacterium  or  red  blood 
cell  which  has  been  affected  by  its  specific  sensitizer  has,  on  the 
contrary,  the  power  of  fixing  a  large  amount  of  alexin,  which  then 
disappears  from  the  surrounding  fluid.  Certain  bacteria,  as  the 
cholera  vibrio,  show  a  morphological  change  as  a  result  of  this 
alexin  fixation  brought  about  by  the  specific  sensitizer;  they  form 
granules  and  then  dissolve.  Sensitized  blood  cells  are  easily  hemo- 
lyzed  by  alexin.  Many  forms  of  bacteria  show  no  marked  mor- 
phological change  through  the  influence  of  sensitizer  plus  alexin. 
But  even  these  bacteria,  when  they  have  been  treated  with  the 
sensitizer,  show  the  property,  as  do  the  more  susceptible  organisms, 
of  fixing  the  alexin.  This  property,  then,  of  fixing  alexin  on  a 
cell  is  generalized  and  fundamental  property  in  every  sensitizer. 
Through  this  property,  Bordet  and  Gengou,f  in  1900,  were  able  to 
demonstrate  the  presence  of  sensitizers  in  certain  active  sera.  These 
authors  showed  that  the  sera  of  animals  that  have  been  immunized 
against  the  typhoid  bacillus,  the  plague  bacillus  and  the  like,  as 
well  as  sera  from  patients  who  have  recently  recovered  from  typhoid 

*  Zur  Kenntnis  der  antituberkulosen  Sensibilisatoren.      Berliner   klin.   Wo- 
chenschr.,  1906,  p.  1531. 
f  Page  217. 

467 


468  STUDIES  IN   IMMUNITY. 

fever,  contains  specific  sensitizers,  inasmuch  as  any  one  of  these 
sera  has  the  property  of  fixing  alexin  in  the  presence  of  the  specific 
organism  against  which  it  was  formed ;  this  property  is  not  possessed 
by  any  one  of  the  sera  under  normal  conditions. 

Since  then  I  have  used  the  same  method*  to  demonstrate  spe- 
cific sensitizers  in  the  serum  of  animals  which  had  received  in- 
jections of  albuminous  substances  such  as  milk,  albumin,  fibrin- 
ogen  or  alien  serum.  An  albuminous  solution  on  the  addition 
of  its  specific  antiserum  acquires  the  property  of  fixing  the  alexin. 

The  technic  employed  is  as  follows :  The  bacteria  or  the  albumin 
in  question  is  mixed  with  a  small  amount  of  alexin  and  the  anti- 
serum  in  which  we  are  endeavoring  to  prove  the  presence  of  a  sen- 
sitizer.  A  few  hours  later  sensitized  red  blood  cells  are  added  to 
this  mixture.  These  cells,  which,  as  we  know,  are  very  susceptible 
to  alexin,  served  to  indicate  whether  the  alexin  which  was  added 
in  the  first  place  has  remained  free  or  been  absorbed  by  the  al- 
buminous bodies.  If  the  bacteria  or  the  albuminous  substance 
has  been  sensitized  by  the  serum  that  is  being  investigated,  it 
absorbs  the  alexin  and  subsequently  added  corpuscles  remain 
intact,  as  no  free  alexin  is  present. 

In  this  communication  I  also  make  use  of  this  method,  which 
was  first  employed  by  Bordet  and  myself  in  1901,  to  demonstrate 
the  presence  of  sensitizers  in  the  serum  of  animals  that  have  been 
immunized  with  various  acid-fast  bacilli  and  for  the  purpose  of 
showing  by  such  sensitizers  the  relations  between  saprophytic 
bacteria  and  those  which  are  pathogenic  either  for  cold-blooded 
or  warm-blooded  animals. 

Very  little  work  has  been  done  with  antituberculous  sensitizers. 
Bordet  and  If  have  been  able  to  show  that  guinea-pigs  immunized 
with  avian  tubercle  bacilli  form  sensitizers  which  are  active  against 
both  human  and  avian  tubercle  bacilli. 

Dembinski  noted  in  1904  that  rabbits  and  doves  which  have  re- 
ceived injections  of  avian  bacilli  form  sensitizers  against  these 
organisms,  but  no  such  sensitizers  following  the  injection  of 
human  tubercle  bacilli.  I  have  questioned  certain  of  this  author's 
conclusions.  $  Wassermann  and  Bruck  §  have  recently  employed 

*  Page  241.  t  See  p.  462.  t  See  p.  464. 

§  Wassermann  and  Bruck,  Deutsche  med.  Wochenschr.,  1906. 


KNOWLEDGE  OF  ANTITUBERCULOUS  SENSITIZERS.         469 

the  Bordet-Gengou  method  to  demonstrate  the  presence  of  anti- 
tuberculous  sensitizers  in  tuberculous  patients  who  have  received 
tuberculin. 

I  have  carried  out  my  experiments  on  guinea-pigs  which  have 
been  immunized  with  the  following  acid-fast  bacilli:  the  homo- 
geneous culture  of  tuberculosis  of  Arloing*,  obtained  from  Lille, 
bacillus  of  fish  tuberculosis,  the  acid-fast  butter  bacilli  of  Rabinow- 
itsch  the  acid-fast  grain  bacilli,  No.  I,  and  Tobler  bacilli,  I,  II  and  V, 
and  also  the  Timothy  bacillus  and  a  bacillus  from  horse  dung.  Cul- 
tures of  these  organisms  were  grown  on  glycerin  potato  without 
any  particular  reference  as  to  their  age  and  were  dried  at  37  degrees 
for  24  hours  in  a  vacuum.  The  bacilli  were  then  ground  up  and 
used  at  various  periods,  subsequently,  for  injection.  Five  milli- 
grams of  powder  shaken  up  in  salt  solution  was  used  for  each  in- 
jection in  the  guinea-pigs.  Three  such  injections  were  given  at 
intervals  of  3  weeks  in  order  to  give  the  animals  time  for  the  ab- 
scesses, which  frequently  follow,  to  heal.  The  guinea-pigs  were 
bled  14  to  21  days  after  the  last  injection  and  the  separated  sera 
as  well  as  the  normal  serum  employed  for  control  were  heated  to 
56  degrees  for  half  an  hour.  Sensitizers  were  then  demonstrated 
by  the  Bordet-Gengou  method,  a  description  of  which  is  given  in 
their  first  article. f  It  may  be  noted  here  simply  that  in  all  these 
experiments  six  parts  of  the  serum  to  be  tested  were  mixed  with  one 
part  of  guinea-pig  alexin  and  two  parts  of  an  emulsion  of  bacilli. 
This  mixture  of  bacilli  is  1  c.c.  in  volume  and  contains  80  milligrams 
of  bacilli  from  a  fresh  culture.^  Three  hours  later  one  part  of 
sensitized  goat  blood  was  added  to  each  tube.§  In  the  following 
table  the  results  of  these  experiments  are  given.  This  table  shows 

*  These  bacteria  were  kindly  given  us  in  part  by  Dr.  Binot  of  the  Pasteur 
Institute,  Paris,  and  in  part  by  Dr.  van  Steenberghe  of  the  Pasteur  Institute  in 
Lille,  to  whom  I  wish  to  express  my  indebtedness. 

f  I  have  demonstrated  to  my  own  satisfaction  that  the  traces  of  potato  which 
of  necessity  must  be  present  in  the  experimental  test  tubes  with  the  bacilli,  or 
which  are  injected  into  the  animal  during  the  immunization  have  no  effect  on 
the  result. 

J  I  have  found  that  it  is  better  to  use  the  fresh  cultures  instead  of  the  dried 
bacilli;  the  dried  powder  frequently  of  itself  absorbs  alexin. 

§  In  all  my  experiments  one  part  of  washed  goat  blood  was  placed  in  contact 
for  one  quarter  of  an  hour  with  one  part  of  heated  immune  serum  and  subsequently 
washed,  and  mixed  with  two  volumes  of  0.85  per  cent  salt  solution. 


470  STUDIES  IN   IMMUNITY. 

that,  in  general,  the  injection  of  acid-fast  bacilli  in  guinea-pigs, 
whether  they  be  saprophytic  or  pathogenic  for  cold-blooded  ani- 
mals, or  of  the  homogeneous  culture  of  Arloing,  gives  rise  to  sen- 
sitizers  which  are  active  not  only  against  the  homologous  bacteria, 
but  also  against  other  acid-fast  bacteria,  whether  saprophytic  or 
pathogenic.  These  sensitizers  are  particularly  active  against  the 
bacilli  of  human,  bovine  or  avian  tuberculosis.  There  are  certain 
exceptions,  however,  to  this  rule  which  are  brought  out  by  my 
experiments.  Acid-fast  Tobler  No.  I  has  no  sensitizer  for  acid- 
fast  Tobler  No.  V;  Bacillus  Tobler  No.  II  has  no  sensitizer  for 
fish  tuberculosis,  and  Tobler  No.  V  none  against  avian  tuberculosis. 
I  have  not  studied  these  exceptions  any  further,  but  have  contented 
myself  with  calling  attention  simply  to  the  general  uniformity  of 
this  immunity  against  tubercle  bacilli,  which  was  formerly  pointed 
out  by  Klemperer  in  guinea-pigs  following  the  injections  of  sapro- 
phytic acid-fast  bacilli. 

It  seems  to  me  wise,  before  drawing  any  conclusions  from  my 
results  as  to  the  relationship  between  the  different  acid-fast  bacilli, 
to  continue  these  experiments  and  particularly  to  follow  more 
closely  the  time  of  occurrence  of  the  antibodies  in  the  immunized 
animals. 


KNOWLEDGE  OF  ANTITUBERCULOUS  SENSITIZERS.        471 


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XXVII.    THE   BACILLUS   OF  WHOOPING-COUGH  * 

BY  DRS.  J.  BORDET  AND  O.  GENGOU. 

The  bacteriology  of  whooping-cough  has  been  studied  to  a  con- 
siderable extent  during  the  last  twenty  years.  Many  bacteria 
have  been  isolated  from  the  sputum  and  described  as  the  true 
etiological  factor  in  this  disease;  we  believe,  however,  that  none  of 
these  micro-organisms  that  have  been  grown  by  our  predecessors 
is  identical  with  the  one  we  have  obtained,  which  latter  organism, 
on  account  of  the  logical  proofs  that  we  have  been  able  to  find, 
must  be  considered  as  the  true  parasite  that  has  been  sought  for. 

The  failure  of  other  bacteriologists,  as  well  as  our  own  failures 
for  nearly  six  years,  during  which  time  we  have  studied  whooping- 
cough,  are  easily  explicable  in  view  of  certain  circumstances  which 
may  be  briefly  mentioned. 

As  every  one  knows,  the  success  of  attempts  to  isolate  an  un- 
known pathogenic  agent  depends  on  the  presence  of  a  sufficient 
number  of  the  organisms  in  the  product  that  is  studied  and  on  the 
fact  that  these  organisms  shall  not  be  lost  in  the  midst  of  innumer- 
able non-specific  bacteria.  These  conditions  of  relative  abundance 
and  purity  of  culture  of  the  specific  organism  are  not  easily  met 
with  in  the  majority  of  cases  of  whooping-cough,  except  in  the 
beginning.  And,  what  is  more,  even  during  this  beginning  period 
only  that  part  of  the  expectoration  which  comes  from  the  region 
in  which  the  bacteria  are  most  active  should  be  used ;  in  other  words, 
the  products  from  the  depths  of  the  bronchi  expectorated  during 
the  periodic  accesses  of  the  disease.  This  exudate  during  the 
characteristic  " whoop"  is  white,  thick,  and  rich  in  leucocytes; 
it  contains  the  bacilli  of  whooping-cough  in  considerable  numbers 
and  in  favorable  cases  in  almost  pure  culture.  At  the  same  time 
the  child  often  expectorates  a  more  transparent  mucous  and  sticky 
secretion  which  contains  less  cells  and  many  fewer  specific  bac- 

*  Le  microbe  de  la  coqueluche.  Annales  de  1'Institut  Pasteur,  vol.  20,  1906, 
p.  731. 

472 


THE  BACILLUS   OF  WHOOPING-COUGH.  473 

teria  and  numerous  other  extraneous  organisms.  This  latter  form 
of  expectoration  should  be  avoided  in  making  cultures.  If  the 
expectoration  is  examined  on  successive  days,  it  is  found  that  the 
leucocytic  exudate  contains  fewer  and  fewer  specific  bacilli  and 
that  phagocytosis  occurs  more  frequently  than  at  first.  Although 
the  accesses  of  whooping-cough  continue  numerous  and  characteris- 
tic, the  richness  of  the  culture  becomes  less.  It  may  be  that  the 
organism  still  persists  in  the  tissue  of  the  bronchi,  but,  at  any  rate, 
it  is  certain  that  the  number  of  organisms  eliminated  by  expecto- 
ration decreases  notably.  This  later  exudate  is  less  suited  for 
growing  the  organisms,  and,  what  is  more  important  still,  micro- 
scopical preparations  of  it  evidence  this  fact  clearly;  to  one  who 
has  studied  the  previous  earlier  expectorations,  the  subsequent  less 
favorable  ones  show  clearly  the  importance  of  this  bacillus  and 
its  function.  The  relatively  short  duration  of  the  favorable  period 
for  obtaining  cultures  of  the  whooping-cough  bacillus,  the  unequal 
distribution  of  the  bacillus  in  the  sputum,"  and,  furthermore,  the 
difficulty  in  obtaining  the  secretions  at  the  proper  moment  from 
infants,  all  render  the  study  difficult.  Although  the  research  is 
difficult  even  when  dealing  with  pure  cases  of  whooping-cough, 
it  becomes  even  more  so  when  the  disease  is  complicated  by  non- 
specific colds,  bronchitis,  bronchopneumonia,  etc.;  in  other  words, 
when  other  bacilli  of  respiratory  affections  are  mixed  with  those 
that  cause  the  disease.  The  sputum  obtained  from  children  at 
home  is  therefore  evidently  much  more  suitable  for  study  than 
materials  obtained  in  the  hospital. 

The  necessary  conditions  for  success  in  such  a  research,  as  well  as 
the  causes  of  error  that  may  throw  an  investigator  off  the  track,  will 
appear  more  clearly  if  we  give  a  short  resume  of  our  own  attempts. 
Incidentally  we  shall  mention  certain  non-specific  bacteria  that  we 
have  frequently  met  with  which  may  be  confounded  with  the  true 
bacillus  and  to  which  other  bacteriologists  have  drawn  attention. 

Our  first  researches  on  whooping-cough  began  in  1900.  At 
this  time  a  child,  B,  aged  5  months,  subsequent  to  contact  with 
children  that  were  beginning  to  cough  and  in  whom  the  diagnosis 
of  whooping-cough  was  later  determined,  suffered  an  attack  of 
typical  whooping-cough.  The  health  of  this  child  had  hitherto 
been  perfect.  She  had  never  had  even  the  slightest  disease  of  the 


474  STUDIES  IN   IMMUNITY. 

respiratory  tract.  One  of  us  who  followed  her  case  carefully  was 
able  to  obtain  a  whitish  shred  of  exudate  that  was  not  mixed  with 
saliva  which  was  projected  by  the  whoop  in  the  first  characteristic 
crisis.  The  microscopical  examination  with  Kuhne's  carbolated 
blue  stain  showed  that  this  rich  leucocytic  exudate  contained  an 
enormous  number  of  little  ovoid  bacilli  which  were  somewhat 
elongated  at  times,  but  frequently  so  short  as  to  resemble  a  micro- 
coccus;  in  general  the  aspect  was  rather  constant,  the  stain  a  pale 
blue,  and  the  outline  of  the  bacillus  and  particularly  its  poles  were 
frequently  more  deeply  stained  than  the  center.  This  organism 
was  found  scattered  indiscriminately  among  the  cells  and  was 
at  times  within  cells.  Some  of  the  larger  organisms  frequently 
showed  a  little  point  toward  the  center  that  appeared  to  be  walled 
off;  the  majority  of  the  bacteria  were  separate,  but  some  of  them 
occurred  in  pairs,  end  to  end.  Gram's  stain  was  negative.  The 
growth  of  the  organism  was  so  abundant  and  the  culture  was  so 
pure  that  it  seemed  reasonable  to  admit  some  causal  relation  be- 
tween this  organism  and  the  appearance  of  whooping-cough  in  a 
child  whose  bronchi  had  not  previously  been  affected.  All  attempts 
to  cultivate  the  organism,  however,  were  unsuccessful.  The  exu- 
date, when  grown  on  ascites-agar  (ascites  fluid  mixed  in  equal  parts 
with  melted  agar)  or  on  agar  moistened  with  human  or  rabbit 
blood,  gave  only  a  few  colonies  of  unimportant  cocci.  Such  media, 
however,  are  very  well  suited  to  grow  delicate  bacteria;  on  blood 
agar,  in  particular,  the  inoculation  of  sputum  from  cases  of  grippe 
gives  an  abundant  growth  of  the  influenza  bacillus.  The  sputum 
on  subsequent  days  showed  a  progressive  diminution  in  this  bacillus. 
In  short,  although  microscopical  examination  showed  many  organ- 
isms, culture  was  unsuccessful. 

During  the  following  years  we  made  a  bacteriological  examina- 
tion of  a  number  of  whooping-cough  sputa  in  Brussels,  the  most 
part  of  which  we  collected  in  hospitals.  We  usually  used  the  cul- 
ture medium  which  we  have  found  very  useful  in  growing  delicate 
bacteria,  particularly  those  bacteria  which  we  have  studied  in  our 
work  on  the  flora  of  the  respiratory  tract.*  The  majority  of  these 

*  The  culture  medium  is  prepared  in  the  following  manner:  100  grams  of  sliced 
potatoes  are  added  to  200  c.c.  of  4  per  cent  glycerinated  water.  This  is  steamed 
in  the  autoclave  and,  on  separating  the  fluid,  a  concentrated  glycerinated  extract 
of  potato  is  obtained.  To  50  c.c.  of  this  extract  is  added  150  c.c.  of  0  per  cent 


THE  BACILLUS  OF  WHOOPING-COUGH.  475 

sputa  were  from  cases  of  whooping-cough  that  were  not  studied  in 
the  beginning,  but  were  well  advanced,  and  they  contained,  there- 
fore, a  rich  and  varied  flora.  We  paid  particular  attention,  ob- 
viously, to  the  study  of  those  bacteria  that  we  had  observed  so 
abundantly  in  a  pure  state  in  the  single  case  in  1900  which  we 
have  mentioned. 

We  found,  indeed,  in  greater  or  less  numbers,  small  ovoid  bac- 
teria which  were  faintly  stained  and  were  quite  similar  to  those 
we  formerly  observed.  The  most  frequent  organism  that  we  found, 
however,  was  still  smaller,  better  colored  and  either  separated 
or  in  clumps  and  frequently  somewhat  long  or  even  filamentous. 
This  organism,  which  occurred  in  almost  every  case  in  abundance, 
grew  well  on  our  medium;  it  gave,  indeed,  in  the  majority  of  cases, 
the  greatest  number  of  colonies.  These  colonies  were  bluish  or 
grayish,  slightly  elevated  in  the  center,  rather  diaphanous,  par- 
ticularly at  the  edges,  and  almost  transparent  in  young  cultures, 
and  resembled  drops  of  dew.  Microscopically  the  organism  was 
small,  did  not  stain  by  Gram,  was  usually  short  and  delicate, 
appeared  frequently  as  a  dot,  and  in  certain  cultures,  particularly, 
showed  a  marked  tendency  to  pleomorphism.  Certain  individual 
bacteria  were  somewhat  thicker  and  swollen  and  others  showed 
curious  involution  forms  which  stained  faintly  or  unequally.  This 
organism,  although  it  does  not  grow  on  agar  or  ordinary  bouillon, 
grows  very  well  in  the  presence  of  hemoglobin.  It  was  easy  to 
determine  that  we  had  to  deal  with  an  organism  which  was  iden- 
tical, or  at  least  analogous,  with  the  one  discovered  by  Pfeiffer 
in  influenza  and  which  other  bacteriologists  have  noted  in  whoop- 
ing cough  and  even  considered  as  the  specific  bacillus — an  opinion 
which  is  reasonable  enough  when  we  consider  its  frequency.  The 
work  of  Jochmann  and  Krause,*  giving  a  very  exact  description 
of  this  bacillus  and  mentioning  its  need  of  hemoglobin,  had  just 

salt  solution  and  5  grams  of  agar.  This  mixture  is  melted  in  the  autoclave  and 
the  warm  fluid  is  placed  in  test  tubes,  2  or  3  c.c.  to  a  tube.  The  tubes  are  then 
sterilized.  Sterile  defibrinated  rabbit  blood,  or  preferably  human  blood,  is  then 
obtained.  To  each  melted  agar  tube  an  equal  amount  of  blood  is  added,  the 
mixture  well  shaken  and  the  tubes  slanted  and  cooled.  This  medium  allows  the 
growth  of  such  delicate  organisms  as  the  meningococcus,  gonococcus,  the  influenza 
bacillus  and,  as  we  shall  presently  see,  the  whooping-cough  bacillus.  As  the  media 
contains  no  pepton  it  is  not  very  good  for  the  growth  of  putrefactive  saprophytes. 
*  Zeitschrift  fur  Hygiene,  1901,  Bd.  36. 


476  STUDIES  IN  IMMUNITY. 

appeared  at  that  time.  This  description,  indeed,  agrees  perfectly 
with  our  own.  We  found  as  well  as  these  authors  that  this  organ- 
ism was  present  in  large  numbers  in  almost  pure  culture  (with  the 
exception  of  a  few  pneumococci)  in  the  pus  of  the  smaller  bronchi 
at  an  autopsy  of  a  child  who  died  of  bronchopneumonia  following 
whooping-cough.  Such  facts  evidently  seemed  significant. 

In  studying  this  problem,  we  naturally  had  to  compare  this 
bacillus  as  it  appeared  in  our  cultures,  or  in  the  newer  cases  which 
we  had,  very  carefully  as  regards  morphology,  with  the  organism 
which  we  found  in  our  first  case  in  1900  and  which  seemed  in- 
contestably  to  be  the  true  parasite.  Such  a  comparison,  however, 
did  not  settle  our  doubt.  This  latter  organism  evidently  belongs  in 
the  group  of  small  bacteria  that  stain  poorly,  as  well  as  the  influ- 
enza bacillus  which  has  already  been  cultivated.  The  appearance 
of  the  two  organisms  is  not,  however,  identical.  If  we  consider 
the  bacteria  that  we  found  in  the  exudate  in  1900  as  typical,  they 
are  of  more  regularly  ovoid  form,  and,  as  a  general  rule,  somewhat 
larger  and  with  a  more  constant  deeply  stained  center.  Might 
it  not,  however,  seem  reasonable  that  a  given  bacillus  in  an  arti- 
ficial medium  should  not  appear  identical  with  one  observed  in  a 
pathological  specimen?  In  short,  the  two  bacteria  might  be  con- 
sidered identical,  and  the  opinion  that  a  bacillus  similar  to  the 
influenza  bacillus  has  an  etiological  function  might  well  be  accepted, 
particularly  when  it  has  been  claimed  by  the  bacteriologists  to 
whom  reference  has  been  made. 

There  are  three  grave  objections,  however,  to  this  hypothesis. 
In  the  first  place  the  bacillus  in  the  typical  exudate  of  1900  did 
not  grow  in  blood  agar,  in  which  medium  the  influenza  bacillus 
would  certainly  have  grown  readily.  Then  we  have  not  been  able 
to  demonstrate  any  particular  properties  against  this  latter  organ- 
ism in  the  serum  of  children  that  have  recovered  from  whooping- 
cough,  in  spite  of  repeated  attempts.  Other  bacteriologists 
must  also  have  obtained  similar  negative  results,  for  no  mention  is 
made  in  the  work  of  Spengler,  of  Jochmann  and  Krause  and  others, 
of  specific  properties  in  the  blood  of  recovered  children.  And 
finally  we  found  that  this  organism  was  present  not  only  in  whoop- 
ing-cough, but  also  in  the  various  respiratory  affections  in  the 
adult  as  well  as  in  children,  in  cases  of  grippe,  bronchitis  and 


THE  BACILLUS  OF  WHOOPING-COUGH.  477 

bronchopneumonia  as  a  complication,  in  various  diseases,  and  also 
frequently  in  a  pure  state  in  simple  coryza.  These  findings  agree 
with  the  facts  of  certain  of  our  predecessors,  notably  with  those  of 
Elmassian. 

In  short,  this  organism,  which  resembles  the  one  described  by 
Pfeiffer  as  the  cause  of  influenza,  is  certainly  not  the  cause  of  whoop- 
ing cough.  We  have  this  year  been  able  to  isolate  a  specific  bacil- 
lus, since  we  have  had  at  our  disposition  favorable  cases,  among 
which  the  one  that  furnished  our  first  successful  culture  may  be 
mentioned  in  some  detail.  It  was  a  male  child  of  two  months, 
nursed  by  its  mother,  in  excellent  health,  who  had  been  contamin- 
ated by  a  neighbor's  child  who  later  on  showed  a  most  characteristic 
case  of  whooping-cough.  The  child  began  to  cough  and  the  charac- 
teristic paroxysms  soon  came;  during  one  of  these  paroxysms  a 
white  bit  of  exudate  which  was  extremely  rich  in  leucocytes  was 
coughed  up.  This  exudate  contained  enormous  numbers  of  a 
micro-organism  which  was  identical  with  the  one  we  had  seen 
several  years  before  and  in  a  similar  condition  of  purity  and 
abundance.* 

This  exudate  was  shaken  up  in  salt  solution  so  as  to  form  various 
dilutions  and  these  dilutions  were  inoculated  on  our  medium.  The 
surface  of  those  tubes  that  had  been  inoculated  with  a  relatively 
concentrated  dilution,  in  which,  therefore,  the  specific  organism 
was  present  in  large  numbers,  showed,  instead  of  a  uniform  growth 
at  the  end  of  2  days,  only  a  few  contaminating  colonies  (some 
coccus  that  occurs  in  the  saliva).  The  platinum  loop,  however, 
removed,  from  the  part  of  the  surface  of  the  culture  which  appeared 
to  be  sterile,  a  large  number  of  the  organisms  that  were  being 
sought  for.  It  appeared  as  if  multiplication  had  actually  taken 
place  but  was  too  slight  to  give  rise  to  visible  colonies,  f  This 
organism,  implanted  on  a  second  tube,  grew  much  better,  giving 
rise  to  a  definite  line  of  implantation,  and  the  culture  then  became 
luxuriant.  Morphologically  the  identity  of  this  organism  with 

*  On  successive  days  the  quantity  of  bacteria  progressively  diminished.  The 
paroxysms  continued  for  about  3  weeks  and  the  child  got  well  after  complica- 
tions. 

t  At  times  we  have  found  that  certain  individual  micro-organisms  give  rise  to 
distinctly  visible  colonies  in  2  or  3  days  even  in  the  first  culture.  This,  however, 
is  the  exception. 


478  STUDIES  IN  IMMUNITY. 

that  present  in  the  exudate  was  not  only  wholly  satisfactory,  but 
as  complete  and  absolute  as  possible.* 

The  cultural  comparison  of  this  organism  with  the  influenza 
bacillus  shows  that  the  two  are  essentially  different.  On  blood 
media  the  whooping-cough  bacillus  grows  much  less  readily  during 
the  first  generation,  but  much  more  luxuriantly  subsequently. 
It  grows  a  little  more  slowly.  Its  growth  is  whitish  and  thicker 
and  without  the  bluish,  diaphanous  appearance  of  the  influenza 
growth.  It  does  not  show  as  great  a  need  of  hemoglobin  as  does 
the  influenza  bacillus,  but  when  grown  subsequently  on  colorless 
ascites-agar  it  develops  in  a  white,  oily,  and  moist  layer  which 
becomes  so  opaque  that  in  2  or  3  days  it  is  almost  as  thick  as 
a  culture  of  typhoid  bacillus  on  ordinary  agar.j  The  whooping- 
cough  bacillus  has  less  tendency  to  pleomorphism  and  involution. 
It  does,  to  be  sure,  become  smaller  during  several  generations  on 
our  blood-culture  medium,  but  it  soon  regains  its  primitive  form 
on  being  inoculated  in  a  liquid  medium  and  then  reinoculated  on 
solid  media.  Although  it  fails  to  grow  on  the  usual  media  steril- 
ized in  the  autoclave,  such  as  agar,  gelatin  and  ordinary  bouillon, 
it  develops  well  in  such  liquid  media  as  1  per  cent  glycerinated 
bouillon  with  equal  parts  of  rabbit  blood  or  serum,  although  under 
these  conditions  its  form  frequently  changes. J 

Although  our  researches  on  the  subject  are  not  finished,  it  is 
probable  that  the  organism  secretes  substances  that  produce  local 
effects  rather  than  general  intoxication,  that  is  to  say,  which  pro- 
voke an  irritating  and  even  a  necrotizing  action.  The  organism 
when  injected  subcutaneously  or  intraperitoneally  in  the  guinea- 
pig  is  fatal  only  in  very  large  doses.  On  injecting  a  little  of  the 

*  We  use,  as  a  stain,  carbolated  toluidin  blue  prepared  in  the  following  manner: 
Toluidin  blue  (Grubler),  5  grams;  alcohol,  100  c.c.;  water,  500  grams;  complete 
sokition  is  allowed  and  then  500  grams  of  5  per  cent  carbolated  water  is  added. 
The  stain  is  filtered  1  or  2  days  later.  This  blue  is  preferable  to  ordinary  carbo- 
lated methylene  blue. 

t  These  remarks  naturally  refer  only  to  the  organism  that  has  been  accus- 
tomed to  growing  on  artificial  media;  it  is  by  no  means  sure  that  the  organism  will 
grow  in  the  first  generation  on  ascites-agar. 

J  This  fact  separates  it  usually  from  the  other  bacteria  that  grow  on  ordinary 
media  noted  by  certain  observers  (Afanassiew,  Czaplewski  and  Henzel,  Vincenzi, 
Manicatide,  Leuriaux,  etc.),  and  are  demonstrable  in  quite  pure  whooping-cough 
sputa.  There  is  no  need  of  considering  these  organisms  in  detail. 


THE  BACILLUS  OF  WHOOPING-COUGH.  479 

whooping-cough  exudate  which  contains  numerous  organisms,  in 
the  eye  of  the  rabbit,  only  very  slight  growth  occurs;  the  aqueous 
humor  remains  limpid  and  white  and  intense  weeping  takes  place, 
together  with  an  excessive  conjunctival  congestion.  Injection 
of  a  small  amount  of  pure  culture  produces  the  same  lesions,  the 
gravity  of  which  is  surprising  in  view  of  the  fact  that  there  is  little 
or  no  multiplication  of  organisms.  If  such  a  course  goes  on  in  the 
bronchi,  it  is  easy  to  understand  the  accesses  and  their  persistence 
even  when  the  growth  of  the  organism  decreases. 

The  authenticity  of  this  bacillus  as  the  causal  agent  in  whooping- 
cough  rests  in  large  part  on  the  circumstances  in  which  it  is  found, 
namely,  the  excessive  growth  of  the  organism  in  pure  culture  during 
the  initial  period  of  diseases  in  young  children  who  have  never 
before  been  ill.  The  principal  argument,  however,  in  favor  of 
this  organism  appears  to  us  to  be  furnished  on  studying  the  specific 
properties  of  the  serum.  The  serum  of  individuals  that  have  never 
had  whooping-cough,  or  who  have  had  it  a  long  time  before,  does 
not  agglutinate  the  bacillus  even  in  low  dilution.  The  sera  of 
children  that  have  recently  recovered  from  the  disease  has  a  mod- 
erate agglutinating  property  which  is  constant  and  evident.  The 
most  remarkable  fact,  moreover,  is  the  intensity  of  sensitizing 
property  in  such  sera.  To  demonstrate  it  we  have  used  our  accus- 
tomed method  of  alexin  fixation. 

The  essentials  of  this  method  which  we  demonstrated  in  1901 
and  employed  to  demonstrate  the  presence  of  sensitizers  in  many 
immune  sera,  are  well  known.*  It  has  since  been  employed  by 
numerous  experimenters  —  Lesourd,  Lambotte,  Fassin,  Cohen,  and 
more  recently  by  Wassermann  and  Bruck.f  One  of  us  established 
in  1900t  that  specific  sensitizers  which  are  active  against  bac- 
teria or  red  blood  cells  endow  the  element  which  they  attack  with 
the  new  property  of  absorbing  alexin  energetically.  If  a  mixture 
of  alexin  (fresh  animal  serum),  of  the  substance  under  considera- 
tion, and  of  a  suitable  sensitizer  (that  is,  immune  serum  heated  to 
56  degrees),  is  prepared  in  suitable  proportions  it  is  found  that  after 
a  certain  period  the  alexin  has  disappeared  from  the  fluid.  Con- 

*  See  p.  217. 

t  The  historical  account  of  our  method,  by  Wassermann  and  Bruck,  of  which 
they  have  made  considerable  use,  is  remarkably  brief. 
{  See  p.  186. 


480  STUDIES   IN  IMMUNITY. 

sequently,  if  sensitized  corpuscles  are  subsequently  introduced  into 
the  mixture,  they  undergo  no  hemolysis.  Certain  other  mixtures, 
the  list  of  which  will  be  found  in  our  previous  articles,  give  the 
necessary  control;  in  one  of  them  in  particular  it  is  proved  that  if 
normal  serum,  which  is  not  sensitizing,  is  used  in  the  first  mixture, 
instead  of  the  specific  serum,  there  is  no  delay  in  the  hemolysis  of 
the  subsequently  added  corpuscles. 

If  we  employ  the  serum  of  children  that  have  recently  recovered 
from  whooping-cough  in  such  a  test  as  this,  the  result  is  very  demon- 
strative. In  our  first  experiment  we  employed  the  serum  of  three 
different  children  who  had  recovered  from  two  weeks  to  a  month 
previously,  and  as  controls  the  sera  of  three  normal  individuals. 

These  different  sera  were  heated  to  56  degrees  and  were  mixed 
in  doses  varying  from  0.1  to  0.3  of  a  cubic  centimeter  with  0.05  or 
0.01  of  a  cubic  centimeter  of  fresh  human  or  guinea-pig  serum  (alexin) 
plus  0.2  of  a  cubic  centimeter  of  an  emulsion  of  the  whooping-cough 
bacillus  (a  24-hour  culture  suspended  in  salt  solution).*  Four 
hours  later,  after  remaining  at  room  temperature,  a  little  well-sen- 
sitized goat  blood  was  added  to  each  tube.  Hemolysis  took  place 
in  a  few  moments  in  the  tubes  containing  normal  serum,  but  the  cor- 
puscles remained  intact,  even  on  the  following  day,  in  those  which 
contained  whooping-cough  serum.  The  sensitizing  property,  then, 
of  the  serum  from  a  case  of  whooping-cough  is  very  energetic  even 
in  so  small  a  dose  as  0.1  of  a  cubic  centimeter.  It  is  scarcely 
necessary  to  mention  that  the  serum  of  such  cases  does  not  fix 
the  alexin  unless  the  whooping-cough  bacillus  is  present;  the  other 
necessary  controls  are  also  made. 

The  organism  resembling  the  influenza  bacillus  acts  no  differ- 
ently with  whooping-cough  serum  than  it  does  with  normal  serum. 
Such  an  experiment  shows  not  only  that  this  latter  organism  has 
nothing  to  do  with  whooping-cough,  but  serves  to  separate  the  two 
bacterial  species  from  one  another,  f  In  other  experiments  the 
same  results  were  obtained.  We  tested  the  sera  from  two  different 

*  The  experiment  may  be  done  with  either  of  these  alexins,  but  the  guinea-pig 
alexin  is,  in  general,  more  favorable  for  hemolysis  and  therefore  better  to  use. 

t  It  should  be  noted  that  the  influenza  bacillus  has  in  itself  a  certain  property 
for  absorbing  alexin  without  the  presence  of  any  sensitizer.  We  have  already 
stated  that  the  whooping-cough  bacillus  does  this  only  when  whooping-cough 
serum  is  present. 


THE  BACILLUS   OF   WHOOPING-COUGH.  481 

children  of  the  same  age  (4  years),  both  of  whom  were  convalescent, 
one  of  whooping  cough  and  the  other  of  bronchopneumonia  follow- 
ing scarlet  fever.  As  a  result,  0.2  of  a  cubic  centimeter  of  the 
serum  in  the  first  case  sensitized  the  whooping-cough  bacillus  and 
produced  alexin  absorption.  The  subsequently  added  sensitized 
corpuscles  remained  intact,  whereas  hemolysis  took  place  in  5 
minutes  in  the  mixture  containing  the  other  serum. 

As  we  regard  the  facts  in  the  etiology  of  whooping-cough  as  well 
established,  we  shall  hope  to  announce  soon  the  results  of  attempts 
at  serumtherapy  or  active  immunization  which  we  have  made.  It 
may  be  mentioned  in  this  connection  that  the  injection  of  a  cubic 
centimeter  of  culture,  killed  by  heating  to  62  degrees,*  produces  no 
unpleasant  symptoms  in  man. 

*  Heating  to  55  degrees  suffices  to  kill  the  organism. 


XXVIII.   AN   ADDITIONAL  NOTE   ON  THE  WHOOPING- 
COUGH    BACILLUS* 

BY  DRS.   J.   BORDET   AND   O.   GENGOU. 

It  has  seemed  to  us  well  to  give  certain  additional  information 
concerning  our  study  of  the  whooping-cough  bacillus  in  order  to 
facilitate  the  work  of  other  bacteriologists  who  are  interested  in 
the  subject. 

Our  researches  during  the  last  year  have  fully  confirmed  our 
conviction  as  to  the  authenticity  of  the  organism  we  described  at 
that  time  as  the  causal  agent  in  whooping-cough.  We  shall  not 
repeat  the  arguments  which  we  gave  in  our  former  article,  but 
will  simply  add  that  we  have  in  every  instance  found  a  strong 
sensitizing  power  in  the  sera  of  children  that  have  recovered  from 
whooping-cough. 

A  culture  medium  which  we  have  already  described  —  a  mixture 
of  rabbit  blood  and  agar  containing  a  little  glycerinated  extract 
of  potato  —  still  seems  to  be  the  best  for  isolating  the  organism. f 
In  growing  the  organisms  the  most  important  fact  is  that  the  whoop- 
ing-cough bacillus,  at  least  in  the  first  culture,  grows  very  slowly; 
at  least  2  days  in  the  thermostat  is  necessary  for  the  appearance 
of  colonies,  and  these  may  remain  very  small  unless  the  medium 
is  most  carefully  prepared.  Little  or  no  growth  takes  place  if  the 
medium  has  become  dried  or  if  enough  contaminating  organisms 
of  rapid  growth  are  mixed  with  the  colonies  and  monopolize  the 
nutrient  properties  of  the  medium.  If  these  unfavorable  con- 

*  Note  cbmple'mentaire  sur  le  microbe  de  la  coqueluche.  Annales  de  1'Institut 
Pasteur,  Vol.  21,  1907,  p.  720. 

t  The  following  observation  is  of  some  importance  in  obtaining  a  growth. 
The  defibrinated  blood  added  to  the  fluid  agar  should  be  very  carefully  mixed 
with  it  by  shaking.  The  blood  is  of  greater  specific  gravity  than  the  agar,  and, 
unless  they  are  carefully  mixed,  the  upper  part  of  the  medium  is  composed  of  agar 
that  contains  very  little  blood,  and  therefore  when  the  tubes  are  slanted  their 
surfaces  offer  a  very  unfavorable  culture  medium. 

482 


ADDITIONAL  NOTE  ON   WHOOPING-COUGH  BACILLUS.       483 

ditions  are  avoided,  colonies  may  be  obtained  even  in  the  first 
culture,  which,  although  not  close  together,  show  a  good  growth  at 
the  end  of  the  third  day,  and  are  distinguishable  by  their  whiteness, 
their  projection  and  their  clearly  circumscribed  outline. 

Certain  sputa  which  are  particularly  favorable,  when  suitably 
diluted  and  inoculated  on  the  culture  medium  have  given  us  almost 
pure  cultures,  including  100  to  200  times  as  many  whooping-cough 
colonies  as  colonies  of  other  bacteria. 

We  have,  as  well  as  other  authors,  mentioned  the  frequency  of 
a  bacterium  similar  to  that  described  by  Pfeiffer  as  the  cause  of  in- 
fluenza, in  the  sputum  of  whooping-cough,*  and  we  have  also  men- 
tioned the  fact  that  the  presence  of  these  organisms  offers  a  serious 
obstacle  to  the  isolation  of  the  true  parasite  of  whooping-cough. 
They  usually  form  numerous  colonies  which  develop  rapidly.  These 
organisms  are  not  agglutinated  by  the  serum  of  a  horse  immunized 
against  the  whooping-cough  bacillus,  although  this  serum  agglu- 
tinates the  latter  organism  energetically;  the  serum  may  therefore 
be  used  as  a  ready  and  infallible  means  of  differentiation.  Micro- 
scopically these  organisms  are  frequently  rather  difficult  to  dis- 
tinguish from  the  real  whooping-cough  organism,  f  A  few  suc- 
cessive cultures  on  blood  media  suffice  to  distinguish  the  two 
organisms  with  certainty.  When  the  whooping-cough  bacillus  is 
inoculated  on  the  surface  of  blood  agar  in  a  delicate  streak,  the 

*  We  might  better  say,  identical  with,  rather  than  similar  to.  We  have  culti- 
vated for  some  time  parallel  cultures  of  bacteria  of  this  sort  coming  from  cases 
of  whooping-cough  and  the  typical  influenza  bacillus  which  was  kindly  given  us 
by  Dr.  Cohen,  who  obtained  it  from  Pfeiffer's  laboratory.  Comparison  of  these 
cultures  shows  no  perceptible  difference  between  the  organisms. 

f  This  is  particularly  true  in  the  first  generation  of  cultures.  Certain  indica- 
tions on  this  point  may  be  useful.  On  suspending  in  water  to  make  a  stained 
preparation,  the  influenza  bacilli  give  an  emulsion  with  a  tendency  to  spontaneous 
agglutination,  so  that  in  drying  on  the  slide  they  are  often  clumped  in  small  masses 
(see,  for  example,  Fig.  1,  plate  9,  in  Jochmann  and  Krause's  article,  Zeitschrift 
fiir  Hygiene,  1901);  in  preparations  of  whooping-cough  the  organisms  are  better 
separated.  After  several  successive  cultures  on  our  medium,  the  influenza 
bacillus  often  assumes  larger  shapes,  which  are  frequently  swollen  and  twisted; 
the  mean  size  increases  and  is  frequently  greater  than  that  of  the  whooping-cough 
organism.  Cabolated  blue  stains  color  the  influenza  bacillus  much  more  intensely 
than  they  do  the  whooping-cough  organism.  We  may  recall  that  transplanting 
on  ascites-agar  serves  as  a  good  method  for  distinguishing  the  two  organisms;  the 
whooping-cough  bacillus  grows  on  it  slowly  as  a  white  streak;  the  influenza  bacillus 
grows  very  slightly,  although  it  does  grow  somewhat. 


484  STUDIES  IN  IMMUNITY. 

growth  of  organisms  thickens,  and  in  2  or  3  days  projects  dis- 
tinctly from  the  surface.  But  although  it  becomes  thicker  it  does 
not  broaden,  so  that  its  sides  are  distinctly  steep.  The  influenza 
bacillus,  when  grown  in  the  same  way,  gives  a  much  wider  streak, 
has  a  festooned  outline  which  slopes  gently,  and  is  moist  and 
glistening.  The  whooping-cough  growth,  moreover,  is  whiter  and 
never  blackens  the  underlying  blood  medium,  whereas  the  influ- 
enza bacillus  frequently  does  so.*  When  the  whooping-cough 
bacillus  is  looked  at  with  transmitted  light  the  growth  of  organ- 
isms appears  as  a  pale  line  which  stands  out  from  the  adjacent 
part  of  the  culture  medium  which  has  not  been  touched  by  the 
inoculation.  This  clarification  of  the  medium  is  due  to  the  fact 
that  the  organisms  have  hemolyzed  the  adjacent  corpuscles  and 
so  diminished  the  opacity  of  the  nutrient  substratum. 

We  have  already  insisted  on  the  fact  that  the  abnormal  forms 
which  occur  frequently  with  the  influenza  bacillus  in  the  form  of 
clubs  or  long  filaments  with  a  tendency  of  swelling,  with  irregularity 
of  aspect  and  poor  staining,  are  rare  in  the  whooping-cough  bacil- 
lus, which  keeps  its  appearance  of  a  small  cocco-bacillus  invariably. 
The  size  of  this  cocco-bacillus,  particularly  in  old  cultures,  may 
be  extremely  reduced.  We  are  speaking  now  of  cultures  on  a 
solid  medium;  the  cultures  on  liquid  media,  which  we  shall 
discuss  in  a  few  words,  show  more  distinct  pleomorphism ;  the 
dimensions  of  the  organisms  become  more  variable,  their  staining 
more  unequal,  and  they  are  frequently  larger  and  less  ovoid  in 
shape. 

Liquid  cultures  grow  well,  provided  we  take  into  account  the 
necessity  of  atmospheric  contact,  which  is  very  marked  in  the 
whooping-cough  bacillus.  We  find,  indeed,  that  the  organism  grows 
only  under  the  best  conditions  of  aerobiosis.  When  grown  in  a 
test  tube  containing  a  suitable  nutrient  fluid  of  several  centi- 
meters in  depth,  and  maintained  in  a  vertical  position,  the  growth 
of  the  organism  is  slow  and  difficult.  When  the  tube  is  placed 
horizontally,  however,  a  cloud  soon  appears,  covering  the  increased 

*  These  influenza  organisms  are  obtained  from  cases  of  whooping-cough  and 
agree  absolutely  in  all  their  characters  with  the  organisms  described  by  many 
of  our  predecessors  and  notably  by  Jochmann  and  Krause.  We  may  recall  that 
for  a  long  time  our  attention  was  attracted  by  these  organisms,  which  are  con- 
stantly present  in  the  sputum  of  whooping-cough. 


ADDITIONAL  NOTE  ON  WHOOPING-COUGH  BACILLUS.       485 

surface.  It  is  therefore  well  to  make  cultures  in  a  flask  with  a 
large  flat  bottom  and  to  employ  not  more  than  a  centimeter  deep 
of  fluid.  An  excellent  culture  medium  is  made  by  a  mixture  of 
1  per  cent  pepton  bouillon  containing  1  per  cent  glycerin  and  equal 
parts  of  horse  serum,  the  latter  preferably  heated  for  three-quarters 
of  an  hour  to  57  degrees.  Under  these  conditions  the  organism  grows 
almost  without  clouding  the  fluid ;  in  4  or  5  days  the  bottom  of  the 
flask  is  covered  with  a  whitish,  slightly  viscous,  and  rather  thick 
sediment.  The  supernatant  fluid  becomes  more  cloudy,  and  the 
deposit  less  coherent  if  rabbit  serum  is  employed  instead  of  horse 
serum,  as  the  latter  has  a  certain  agglutinating  property  for  the 
organism. 

If,  instead  of  using  normal  horse  serum  (57  degrees)  for  the 
culture  medium,  we  use  the  serum  of  a  horse  that  has  been  im- 
munized against  the  organism  (also  heated  to  57  degrees),  growth 
still  takes  place.  The  bacteria,  however,  are  more  agglutinated 
and  show  a  more  abnormal  appearance;  they  grow  in  the  form  of 
strepto-bacilli,  or  may  even  appear  as  streptococci  in  long  chains, 
their  appearance,  in  short,  being  quite  abnormal  and  resembling 
in  no  particular  the  morphology  on  solid  media. 

Immunization  of  a  horse  gives  rise  to  an  extremely  agglutinating 
serum.*  If  a  two-day  growth  of  culture  on  blood  agar  is  sus- 
pended in  2  or  3  c.c.  of  salt  solution,  and  the  organisms  allowed  to 
diffuse  in  the  suspension  without  much  agitation,  we  obtain  a  cloudy 
fluid  of  homogeneous  and  almost  colloidal  appearance.  This  emul- 
sion is  agglutinated  by  traces  of  the  active  serum;  for  example,  agglu- 
tination takes  place  on  the  addition  of  0.002  of  a  cubic  centimeter  of 
serum  to  1  c.c.  of  the  emulsion.  Too  great  an  excess  of  agglutinin 
inhibits  the  phenomenon;  the  whooping-cough  bacillus,  indeed, 
may  serve  as  a  good  example  to  study  this  inhibiting  power  of 
an  excess  of  serum,  which  has  already  been  noted  by  various 
observers. 

The  various  strains  of  whooping-cough  bacilli  obtained  from 
different  cases  of  the  disease  agglutinate  variably.  We  find,  for 
example,  that  our  horse  serum  agglutinates  the  organism  that  was 

*  Our  horse  received  in  15  successive  injections,  for  the  most  part  subcutane- 
ously,  but  some  intravenously,  a  total  of  about  two  liters  and  a  half  of  rich  fluid 
culture  (glycerinated  bouillon  and  horse  serum) . 


486  STUDIES  IN   IMMUNITY. 

used  to  immunize  the  animal  less  than  it  does  another  organism 
from  a  different  case  of  whooping  cough. 

We  have  hoped  to  be  able  to  make  a  serum  diagnosis  of  whoop- 
ing cough  by  the  simple  and  practical  agglutination  method.  We 
have  found,  unfortunately,  that  the  serum  of  children  suffering 
from  or  convalescent  of  whooping  cough  shows  very  inconstant 
agglutinating  properties.  An  agglutinating  property  is  frequently 
distinctly  manifest,  although  never  very  intense;  it  is  often,  how- 
ever, entirely  lacking.  There  are  many  sera  which,  although  not 
agglutinating,  are  distinctly  sensitizing.  As  we  have  already  men- 
tioned, the  alexin-fixation  method  always  gives  very  marked  positive 
results.  The  two  properties  of  agglutination  and  fixation  are  there- 
fore quite  separate  in  this  disease.  In  this  connection  we  may 
recall  that  the  serum  from  the  convalescent  case  of  typhoid  fever, 
with  which  we  made  our  first  attempts  at.alexin  fixation  (1901), 
was  also  strongly  sensitizing  without  being  agglutinating,  and  we 
have  frequently  noted  this  fact  since  that  time.  Various  other 
observers  have  also  noted  the  lack  of  necessary  parallel  between 
agglutination  and  bactericidal  power. 

We  have  already  noted  the  lesions  (excessive  irritation  and  cloud- 
ing of  the  cornea)  which  follow  the  injection  of  a  sputum  containing 
the  organism  in  pure  state,  or  of  a  culture  of  the  organism  in  the 
anterior  chamber  of  the  rabbit's  eye.  Certain  remarkable  phenom- 
ena are  also  to  be  noted  on  injecting  the  organism  in  the  guinea-pig's 
peritoneal  cavity,  and  similar  effects  take  place  in  the  rabbit's  peri- 
toneal cavity  if  larger  doses  are  employed.  For  this  purpose  an 
emulsion  of  culture  from  blood  agar  2  or  3  days  old  is  better  than 
the  less  virulent  fluid  cultures.  1.5  to  2  milligrams  of  bacteria, 
weighed  in  a  moist  condition,  causes  death  on  the  following  day 
or  on  the  second  day.  We  have  to  deal  here,  not  with  an  infection, 
for  the  bacteria  show  no  marked  increase  in  the  peritoneal  cavity; 
at  autopsy  only  a  few  of  the  organisms  are  found  in  the  exudato, 
and  frequently  all  of  them  are  phagocyted  by  the  large  number  of 
leucocytes  present.  Intoxication  phenomena,  however,  are  very 
marked,  and  are  evidenced  by  the  appearance  of  petechiae  in  the 
peritoneal  wall,  and  also  at  times  in  the  pericardial  cavity,  and 
by  an  extreme  congestion  of  the  cardiac  blood  vessels.  Fatty 
degeneration  of  the  liver  is  also  found.  The  most  apparent  symp- 


ADDITIONAL  NOTE  ON  WHOOPING-COUGH  BACILLUS.       487 

torn  that  the  animal  shows  before  death  is  marked  dyspnoea,  which 
appears  very  early  and  may  be  accounted  for  by  the  pleural  exu- 
date.  A  subcutaneous  injection  causes  an  edema. 

An  emulsion  of  culture  suspended  in  this  way,  and  then  killed 
by  toluol  or  by  heating  to  56  degrees  for  half  an  hour,  when  in- 
jected into  the  peritoneal  cavity  of  guinea-pigs,  also  causes  death, 
with  phenomena  of  intoxication  and  particularly  with  pleural 
exudation.  It  does  not,  however,  give  rise  to  petechiae  and  larger 
doses  are  necessary  than  of  the  living  organism.  The  serum  of 
the  immunized  horse,  although  strongly  agglutinating,  has  only 
slight  antitoxic  effect.  Very  large  doses  are  necessary  to  neu- 
tralize the  bacterial  emulsion  so  that  it  may  be  injected  into  the 
peritoneal  cavity  without  producing  injury.  It  may  be  added 
that  we  have  only  a  single  immunized  horse,  and  therefore  we 
must  be  somewhat  reserved  in  speaking  of  the  properties  of  the 
immune  serum,  as  these  properties  may  be  due  to  the  individual 
animal  employed.  It  is,  however,  very  likely  that  horses  immu- 
nized against  the  whooping-cough  bacillus  are  similar  to  those 
that  furnish  antityphoid  serum,  the  serum  of  which,  although 
showing  certain  properties,  such  as  agglutination  very  markedly, 
are  not  extremely  antitoxic.  This  latter  property  we  shall  endeavor 
to  increase.  The  results  obtained  by  treating  children  suffering 
from  whooping-cough  with  the  serum  in  question,  although  at  times 
distinctive,  have  not  yet  been  sufficiently  marked. 


XXIX.    THE   ENDOTOXIN   OF  THE  WHOOPING-COUGH 

BACILLUS.* 

BY  DR.   BORDET. 

In  the  recent  studies  on  whooping-cough  with  Dr.  Gengou  we 
have  succeeded  in  obtaining  an  endotoxin  from  the  organism  which 
we  isolated  some  time  ago,  and  which  is  the  cause  of  this  disease. 

We  already  possess  considerable  knowledge  concerning  the  endo- 
toxins  of  bacteria.  Certain  bacteria  diffuse  the  poisons  that  they 
form  readily,  as,  for  example,  the  tetanus  bacillus  and  B.  diph- 
therisc;  other  bacteria,  as  the  typhoid  bacillus,  retain  the  toxin 
within  their  bodies.  Certain  particular  proceedings,  then,  are  neces- 
sary to  remove  these  active  substances  from  the  bacterial  bodies. 
No  absolute  line  of  demarcation  can  indeed  be  drawn  between 
diffusible  toxins  and  endotoxins,  and  there  are  all  degrees  in  the 
ease  with  which  the  poisons  leave  the  bacterial  bodies  and  appear 
in  the  surrounding  fluid,  depending  on  the  organism  under  con- 
sideration. 

In  immunizing  animals  with  an  endotoxin,  it  is  desirable  to 
obtain  it  in  a  clear  solution  without  the  presence  of  the  bacterial 
bodies  themselves.  In  many  instances  it  is  of  importance  to 
immunize  against  the  poison,  particularly,  and  the  presence  of  the 
bacterial  bodies  themselves  gives  a  useless  and  often  unpleasant 
complication.  Too  great  amounts  of  bacteria  wear  out  the  animals, 
lead  to  suppuration,  and,  what  is  more,  the  immunity  against  the 
toxin  is  more  easily  obtained  when  the  bodies  that  form  it  are  not 
present;  it  would  seem  to  diffuse  better  throughout  the  animal 
body  and  to  stimulate  more  those  cells  which  have  to  do  with  the 
elaboration  of  antitoxins.  For  example,  we  find  in  the  case  of 
the  typhoid  bacillus  that  a  subcutaneous  injection  of  the  bacteria 
themselves  produces  no  anti-endotoxin;  the  endotoxin  must  first 

*  L'endotoxine  coquelucheuse.  Bulletin  de  la  Socie'te'  Royale  des  Sciences 
M&licales  et  Naturelles  de  Bruxelles,  No.  7,  1908. 

488 


ENDOTOXIN  OF   WHOOPING-COUGH   BACILLUS.  489 

of  all  be  separated  in  order  to  produce  its  antibody.  In  previous 
articles  we  have  insisted  on  the  fact  that  the  rapid  death  of  guinea- 
pigs  that  have  received  an  injection  of  a  sufficient  number  of 
living  whooping-cough  bacilli  in  the  peritoneal  cavity  is  due  to  an 
intoxication  from  the  poison  that  comes  from  the  bacteria,  and 
not  to  an  infection,  properly  speaking.  The  bacteria  injected  do 
not  increase  to  any  extent  in  vivo,  and  do  not  invade  the  animal 
body;  they  are  retained  at  the  point  of  inoculation,  and  are  fre- 
quently phagocyted.  At  autopsy,  however,  considerable  lesions 
are  found  and  in  particular  a  marked  peritoneal  exudate  of  hemor- 
rhagic  character,  abundant  disseminated  ecchymoses,  and  pleural 
effusions  which  are  so  extensive  as  to  produce  asphyxia,  and  are 
the  immediate  cause  of  death.  All  these  phenomena  may  be  pro- 
duced by  almost  perfectly  limpid  extracts  containing  endotoxin 
which  we  have  obtained  by  employing .  Besredka's  method  as 
described  for  typhoid,  plague  and  dysentery  endotoxins.  We 
inoculate  the  whooping-cough  bacillus  on  our  blood-agar  medium, 
and  in  making  this  medium  we  prefer  horse  blood  in  order  to  obtain 
large  amounts  of  media  readily;  we  leave  the  cultures  in  the  incu- 
bator for  3  days,  and  then  remove  the  layer  of  bacteria  with  a 
glass  rod  and  suspend  them  in  a  small  amount  of  salt  solution.  The 
very  thick  emulsion  obtained  in  this  way  is  dried  in  a  vacuum  at 
37°  C.  in  the  presence  of  caustic  potash.  The  dried  bacteria  are 
then  ground  in  a  mortar  with  the  addition  of  a  little  dried  sterilized 
salt.  Enough  distilled  water  is  then  added  to  this  powder  to  bring 
the  tonicity  of  the  solution  to  normal  (0.75  per  cent).  This  emul- 
sion is  left  until  the  following  day,  energetically  centrifugalized, 
and  the  supernatant  fluid  which  is  decanted  is  almost  limpid. 

This  fluid,  so  far  as  suspended  materials  are  concerned,  contains 
sodium  chloride  in  relatively  large  amount  and  bacterial  substance 
in  proportionately  small  amount.  And  yet  this  fluid  on  intraperi- 
toneal  injection  kills  a  guinea-pig  in  doses  of  0.5  and  even  0.25  of  a 
cubic  centimeter,  with  the  phenomena  that  we  have  just  mentioned. 

Intravenous  injection  in  a  rabbit  of  1  or  2  c.c.  of  endotoxin  is 
usually  fatal,  killing  in  less  than  24  hours.  At  autopsy  renal 
hemorrhages,  acute  fatty  degeneration  of  the  liver  and  a  marked 
hemorrhagic  condition  of  the  suprarenal  capsules  are  found.  In 
respect  to  this  latter  symptom,  as  well  as  in  its  tendency  to  pro- 


490  STUDIES  IN  IMMUNITY. 

duce  pleural  effusions,  the  whooping-cough  toxin  shows  certain 
analogies  with  diphtheria  toxin. 

The  most  interesting  observations,  however,  are  furnished  on 
subcutaneous  inoculation.  Guinea-pigs  that  have  received  an  injec- 
tion under  the  skin  of  0.5  and  even  0.25  of  a  cubic  centimeter  of 
endotoxin  show  a  very  marked  edema,  which  frequently  becomes 
hemorrhagic  at  the  point  of  inoculation;  the  surrounding  area 
rapidly  takes  a  dark  color  without  any  tendency  to  suppuration. 
A  rapid  extensive  necrosis  of  the  skin  follows  and  breaks  down, 
leaving  a  large  ulcer.  This  local  effect  is  much  more  marked  than 
are  the  generalized  symptoms  on  subcutaneous  inoculation;  in 
horses,  ulcers  a  decimeter  square  may  be  produced ;  a  marked  wast- 
ing is  always  noted. 

The  demonstration  of  these  properties  in  the  whooping-cough 
toxin  seems  to  us  to  round  out  satisfactorily  the  ideas  which  we 
previously  had  as  regards  the  pathogenesis  of  whooping-cough. 
The  production  by  the  bacillus  of  an  extremely  irritating  poison 
which  is  capable  of  producing  necrosis  of  the  epithelial  lining  of 
the  bronchi  where  the  organism  multiplies  will  explain  the  charac- 
teristic symptoms  of  the  disease  and  the  appearance  and  persist- 
ence of  the  violent  accesses. 

The  fact  that  whooping-cough  is  not  generally  a  very  serious 
disease,  in  spite  of  the  toxic  power  of  the  causative  bacillus,  is 
easily  understandable  in  view  of  the  fact  that  the  organism  does 
not  tend  to  produce  a  generalized  infection. 

The  whooping-cough  bacillus  is  unfortunately  rather  difficult 
to  handle  on  account  of  its  remarkable  instability  on  heating,  which 
prevents  sterilizing  it.  The  grinding  up  of  the  bacteria  is  neces- 
sarily done  in  the  open  air,  and  it  is  impossible,  therefore,  to  avoid 
dust  and  consequently  contamination  of  the  toxin.  Steriliza- 
tion such  as  is  employed  to  eliminate  contaminating  organisms 
rapidly  destroys  our  toxin,  or  at  least  weakens  it  very  markedly. 
On  heating  to  55  degrees  for  a  short  time  the  endotoxin  solution 
becomes  opaque  and  almost  inoffensive. 

The  use  of  chloroform,  toluol  and  thymol,  which  ordinarily  arc 
so  useful  in  conserving  toxins  without  harming  them,  weaken  the 
whooping-cough  toxin  very  much,  and  render  it  almost  useless. 
We  have  been  forced,  therefore,  to  keep  our  bacilli  ground  with 


ENDOTOXIN  OF  WHOOPING-COUGH  BACILLUS.  491 

salt  in  a  dry  condition,  and  to  add  distilled  water  just  before  using 
them.  In  this  way  the  poison  may  be  kept  for  some  time,  although 
we  have  noted  that  even  when  kept  from  moisture  it  slowly  loses 
its  power. 

The  serum  that  we  formerly  obtained  by  injecting  animals  with 
living  cultures  has  no  power  to  neutralize  the  endo toxin.  We  are 
now  engaged  in  an  attempt  to  obtain  an  antitoxic  serum  by  in- 
jecting endotoxin  instead  of  bacteria. 


XXX.    RESEARCHES   ON   AVIAN   DIPHTHERIA.* 

BY  DR.   BORDET. 

Dr.  Fally  and  I  have  recently  been  studying  the  diphtheria  of 
hens.  This  disease  has  long  interested  bacteriologists  on  account 
of  certain  analogies  that  it  shows  to  human  diphtheria.  Although 
the  acute  and  rapid  course  of  the  disease  in  man  differs  from  that 
in  fowls,  where  it  is  slow  in  evolution  and  chronic,  it  was  formerly 
believed  that  the  relation  between  the  two  diseases  was  very  close. 
This  supposed  relation  was  formerly  frequently  used  to  explain 
epidemics  of  human  diphtheria  as  of  avian  origin.  This  hypothe- 
sis, however,  has  not  been  borne  out  by  the  pathologist. 

The  bacillus  which  causes  human  diphtheria  is  not  met  with  in 
these  hens.  Nor  does  antidiphtheritic  serum,  as  Gratia  and  Lienaux 
have  shown,  have  any  effect  as  a  preventive  or  a  cure  in  avian 
diphtheria.  The  specific  bacteria  in  these  two  diseases  are  cer- 
tainly different. 

Many  organisms  have  been  described  as  causing  the  diphtheria 
in  hens  and  pigeons.  It  is  to  be  noted,  however,  that  none  of  these 
organisms  that  have  been  mentioned  produces  the  disease  with  its 
slow,  capricious,  and  irregular  course,  nor  the  lesions  which  dis- 
appear in  one  place  and  reappear  in  another,  and  show  a  gravity 
and  duration  that  varies  with  the  individual.  Avian  diphtheria 
has  not,  in  the  majority  of  cases,  the  appearance  of  a  disease  which 
tends  to  generalize  and  become  a  septicaemia. 

The  technic  that  has  been  employed  by  many  observers  lias 
been  of  a  nature  designed  to  bring  out  organisms  that  cause  sep- 
ticaemias. Those  who  have  studied  it  have  frequently  inoculated 
healthy  animals  with  false  membranes.  At  times  a  generalized 
infection  was  produced  and  an  organism  isolated  at  autopsy  from 
the  heart's  blood.  The  chances  are,  in  such  cases,  that  the  organ- 

*  Recherches  sur  la  diphte*rie  aviaire.  Bulletin  public*  par  la  Socie"te"  royale 
des  sciences  me"dicales  et  naturelles  de  Bruxelles,  June  3,  1907. 

492 


RESEARCHES   ON  AVIAN  DIPHTHERIA.  493 

ism  obtained  was  the  cause  of  an  accessory  infection,  and  simply 
associated  with,  but  not  the  specific  cause  of,  the  disease. 

It  has  seemed  to  us,  therefore,  necessary  on  account  of  the  ten- 
dency of  the  disease  to  remain  localized,  to  isolate  the  organism 
from  the  morbid  product  itself  rather  than  attempt  a  purification 
of  culture  in  the  animal  body.  A  microscopical  examination 
of  a  bit  of  false  membrane  sufficed,  however,  to  convince  us  that 
this  product  is  very  unsuitable  for  purposes  of  direct  isolation. 
The  membrane,  indeed,  is  swarming  with  different  bacteria,  the 
separation  of  which  would  be  a  difficult  and  probably  fruitless 
task.  The  buccal  cavity  contains,  as  we  know,  under  normal 
conditions,  many  bacterial  species,  and  these  ordinary  organisms 
are  still  more  increased  when  lesions  are  present.  It  seemed  better, 
then,  to  produce  the  specific  infection  in  some  region  of  the  body 
which  is  better  protected  from  saprophytes,  and  the  nictitating 
membrane  in  the  hen's  eye  seems  to  answer  this  requirement.  A 
thread  that  has  been  wet  in  water  in  which  a  fragment  of  false 
membrane  has  been  suspended  is  passed  through  the  nictitating 
membrane.  On  the  following  day  this  thread  is  withdrawn,  and 
the  irritation  due  to  trauma  disappears.  Three  or  four  days  later 
a  grayish  point  on  the  nictitating  membrane  appears  at  the  point 
of  inoculation. 

This  point  then  becomes  red  and  thickened,  and  soon  shows  a 
typical  diphtheritic  lesion;  a  marked  edema  is  also  noticeable  about 
the  eye.  At  this  time,  that  is,  about  the  eighth  day,  the  nictitating 
membrane  is  cut  out  and  washed  in  sterile  salt  solution,  and  then 
ground  up  in  a  few  drops  of  the  same  solution.  The  suspension 
obtained  in  this  way  is  inoculated  on  the  glycerinated-potato-blood- 
agar  medium  which  Gengou  and  I  have  employed  in  growing  the 
whooping-cough  bacillus.  This  suspension  when  inoculated  on 
the  buccal  membrane  of  a  normal  hen  produces  typical  diphtheria. 
The  surprising  thing  is  that  microscopical  examination  with  a 
stain  of  carbolated  toluidin  blue  or  with  the  Giemsa  stain  shows 
no  definite  bacteria;  a  few  granulations,  and  at  times  somewhat 
elongated  spots,  are  seen,  which  are  so  small  as  not  to  be  distin- 
guishable from  cell  debris.  The  culture  tubes  inoculated  with  this 
emulsion  are  incubated  for  3  or  4  days.  Only  a  very  small  num- 
ber of  colonies  is  evident  after  this  period;  and  these  colonies 


494  STUDIES  IN  IMMUNITY. 

on  account  of  their  rarity  are  obviously  due  to  contaminating 
organisms.  It  would  seem  that  the  surface  of  the  culture  medium, 
which  had  been  smooth  and  shiny,  was  uniformly  roughened  be- 
tween these  colonies,  but  the  change  is  so  very  slight  that  it  might 
easily  be  overlooked.  Even  with  a  magnifying  glass  a  layer  of 
bacterial  growth  could  not  be  asserted  to  be  present.  But  if  a 
drop  of  water  is  inoculated  with  a  scraping  from  this  surface,  with 
great  care  in  avoiding  the  colonies  of  contaminating  organisms, 
and  this  water  used  to  inoculate  the  buccal  mucosa  of  a  normal 
hen,  the  disease  is  produced. 

This  same  fluid  on  microscopical  examination  shows  an  enormous 
number  of  little  granular  elements  which  are  at  times  slightly  elon- 
gated about  0.0002  of  a  millimeter  in  size,  and  are  most  frequently 
united  in  compact  zooglea  masses. 

The  culture  may  be  transplanted  and  retransplanted  indefinitely. 

The  layer  of  bacterial  growth  that  is  formed  is  never  thick  enough 
to  be  very  evident.  It  is  evidenced  simply  by  a  darkening  and 
roughening  of  the  surface.  At  times,  however,  very  luxuriant 
cultures  are  obtained  which  show  extremely  small  colonies  or  a 
distinct  outline  limiting  the  area  covered  with  growth.  The  Giemsa 
stain  is  the  best  to  demonstrate  the  organism. 

This  organism,  together  with  the  one  causing  peri-pneumonia 
of  cattle,  is  probably  the  smallest  that  has  ever  been  grown.  It 
generally  reproduces  the  disease  in  a  somewhat  attenuated  form, 
to  be  sure,  particularly  when  the  buccal  mucosa  is  inoculated. 
The  white  plaques  that  appear  in  2  or  3  days  often  heal  rapidly, 
but  also  frequently  reappear  later  in  the  same  place,  and  then 
become  more  extensive.  These  lesions,  although  somewhat  benign, 
are  of  typical  and  undoubted  specificity. 

Certain  hens  are  refractory.  Inoculation  on  the  nictitating 
membrane  is  always  severe  and  always  followed  by  characteristic 
symptoms,  such  as  edema  of  the  eye,  and  thickening  and  redness 
of  the  third  eyelid,  which  shows  the  chronic  nature  of  the  disease. 
To  demonstrate  our  infecting  experiment  on  the  nictitating  mem- 
brane more  conclusively,  and  to  avoid  any  objection  that  this 
infection  is  simply  due  to  a  contagion,  or  a  natural  propagation 
of  the  disease  in  the  laboratory,  and  not  an  artificial  infection,  we 
have  passed  a  thread  through  each  nictitating  membrane  in  a  nor- 


RESEARCHES   ON  AVIAN  DIPHTHERIA.  495 

mal  hen.  One  of  these  threads  was  wet  with  a  culture,  and  the 
other  was  sterile;  on  the  following  day  these  threads  were  drawn 
out.  The  disease  appeared  only  in  the  eye  that  received  the  in- 
fected thread.  We  have  hitherto  dealt  only  with  the  diphtheria 
in  hens,  but  we  propose  to  study  a  similar  disease  found  in  pigeons 
very  soon,  and  to  endeavor  to  determine  whether  it  is  due  to  the 
same  organism  as  the  one  described. 


XXXI.     A  GENERAL   RESUME   OF   IMMUNITY. 

BY  PROFESSOR  JULES  BORDET. 

THE  majority  of  the  articles  which  my  friend  and  collaborator, 
Dr.  Gay,  has  been  so  kind  as  to  translate  and  bring  together  in  this 
volume,  bear  on  the  general  subject  of  immunity  and  have  all  more 
or  less  a  single  trend.  The  logical  connection  between  these  articles 
will  be  quite  evident  to  the  reader,  who  will  have  grasped  my 
general  conception  of  the  mechanism  of  immunity  and  of  the  mode 
of  action  of  sera  so  far  as  we  can  understand  them  in  the  present 
stage  of  development  of  science.  I  may  content  myself,  then,  in 
this  final  chapter  with  certain  additional  explanations. 

I  should  have  been  pleased  to  conclude  this  volume  with  a  syn- 
thetic view  of  the  entire  subject,  or  a  general  theory  capable  of 
coordinating  the  many  facts  that  have  been  acquired.  But  in 
spite  of  the  results  that  have  been  obtained  by  an  army  of  investi- 
gators for  many  years,  I  must  admit  that  such  an  attempt  seems  to 
me  at  the  present  day  both  rash  and  of  questionable  value.  It  is 
rash,  on  account  of  the  great  gaps  that  still  exist  in  our  knowledge 
of  immunity.  In  spite  of  the  numerous  data  which  we  possess,  it  is 
as  yet  impossible  to  offer  a  coherent  whole  or  an  harmonious  and 
complete  system;  many  of  the  facts  which  we  have,  cannot,  as  yet, 
be  classed  according  either  to  relations  or  consequences.  Anyone 
who  should  attempt  at  the  present  day  to  penetrate  the  mystery 
which  shrouds  the  numerous  problems  of  immunity  by  reasoning 
alone  would  be  sure  to  fall  into  error.  Immunity,  like  other  bio- 
logical sciences,  does  not  permit  overgeneralizing  and  adventurous 
theories.  Immunity  is  scarcely  in  a  shape  to  permit  profitable 
deduction,  since  deduction  in  its  endeavor  to  penetrate  too  rapidly 
and  deeply  into  the  unknown,  loses  immediate  contact  with  estab- 
lished facts.  Are  not  discoveries  frequently  unexpected?  Do  not 
experimenters  find  that  their  researches  overthrow  expectations 
which  seem  quite  reasonable,  and  is  it  not  evident  that  logic  totters 

496 


A  GENERAL  RESUME   OF   IMMUNITY.  497 

when  it  would  force  facts?  What  is  perhaps  the  most  obscure  and 
important  question  with  which  immunity  confronts  us?  Evidently 
the  question  of  the  specificity  of  immunization  as  shown  in  the 
specificity  of  sera.  The  solution  of  this  question,  however,  is 
probably  wholly  unsuspected.  Anyone  who  should  attempt  to 
furnish  a  solution  at  the  present  moment,  or  to  give  a  distinctive 
explanation  as  a  result  of  reasoning  on  the  subject,  would  neces- 
sarily be  led  to  support  it  by  undemonstrated  facts.  It  is  better, 
then,  to  seek  for  the  truth  without  wishing  to  define  it  before  we 
have  found  it. 

My  objection  to  too  generalized  conceptions  do  not  in  the  least 
prevent  me  from  recognizing  the  ingenuity  and  the  genius  of  those 
who,  like  my  esteemed  colleague  Ehrlich,  have  proposed  a  general 
interpretation  of  specificity.  Ehrlich 's  theory  has  influenced  too 
many  minds,  has  become  too  generally  known  on  account  of  the 
well  merited  reputation  of  its  author,  not  to  deserve  thorough  con- 
sideration. My  own  impression  of  the  theory  has  not  been  favor- 
able, and  it  has  seemed  my  duty  to  combat  it.  Its  principal  fault 
to  my  thinking  is  that  it  is  not,  strictly  speaking,  a  theory,  but 
rather  an  assertion  of  a  certain  number  of  undemonstrated  facts. 
According  to  Ehrlich,  antibodies  are  produced  as  follows:  in  the 
first  place  the  antigen  when  introduced  into  the  animal  body  meets 
with  a  substance  with  which  it  unites.  So  far  we  all  agree.  It  is 
quite  certain  that  foreign  substances  which  lead  to  the  formation 
of  antibodies  are  taken  up  by  the  tissues  and  produce  a  reaction 
with  certain  substances  in  the  body  to  which,  for  convenience,  the 
name  of  receptors  may  be  given.  The  body  restores  these  destroyed 
receptors  by  producing  new  ones  identical  with  the  original.  But? 
according  to  Ehrlich,  these  new  receptors  are  over-produced  to  such 
an  extent  that  they  flow  over  into  the  fluid  of  the  body,  retain  their 
essential  property  of  uniting  with  the  antigen,  and  are  then  to  be 
designated  as  the  antibodies  of  the  serum.  In  short,  the  antibody 
is  identical  with  the  receptor  affected  by  the  antigen.  To  draw 
such  a  conclusion  is,  however,  to  affirm  a  fact  that  has  never  been 
demonstrated.  Wholly  different  hypotheses  might  as  legitimately 
be  offered.  We  might  suppose,  for  instance,  that  the  body  of  the 
animal  that  is  being  immunized,  instead  of  reproducing  old  recep- 
tors in  large  amount  without  changing  them,  builds  up  substances 


498  STUDIES  IN  IMMUNITY. 

which  in  their  character  and  cellular  appearance  resemble  but  are 
not  completely  identical  with  pre-existent  principles.  These  new 
substances,  as  a  result  of  elaboration  and  change,  have  become 
endowed  with  special  properties,  notably  a  more  marked  affinity 
for  the  specific  antigen  in  question.  In  view  of  the  wonderful 
faculty  of  adaptation  of  organisms  and  their  inexhaustible  resources, 
in  view,  for  example,  of  what  one  sees  (as  Landsteiner  and  Reich 
have  done)  in  Oscillaria  that  have  been  submitted  to  the  influence 
of  certain  luminous  rays  and  which  take  specifically  the  comple- 
mentary color  to  that  possessed  by  these  rays  (Engelman  and 
Gaidukow),  such  an  hypothesis  would  be  in  no  way  irrational. 
Both  conceptions,  then,  are  a  priori  equally  possible  to  defend. 
Why  should  we  choose  one  of  them  and  condemn  the  other  before 
awaiting  the  result  of  experimentation?  Such  solutions  given 
prematurely  to  obscure  problems  are  all  the  more  useless,  as  with 
all  their  apparent  detail  they  fail  to  answer  definite  important 
questions.  In  fact  these  over-produced  receptors  are  but  vague 
names.  Where  are  they  to  be  found?  Are  they,  before  immuni- 
zation, enclosed  within  cell  protoplasm  or  do  they  swim  freely  in  the 
blood?  More  exactly,  we  are  aware  that  in  the  blood  of  normal 
animals  there  are  normal  antibodies,  the  protecting  power  of  which 
is  in  general  slight  but  nevertheless  detectable,  such  substances  as 
agglutinins,  sensitizers  and  even  antitoxins.  It  is  just  as  logical  to 
suppose  that  bacteria  or  poisons,  when  injected  into  a  normal 
animal,  react  with  these  normal  antibodies.  Are  these  normal 
antibodies  really  the  same  receptors  which  are  subsequently  over- 
produced to  form  the  specific  antibody  that  is  characteristic  of  the 
immunity  that  has  been  acquired?  Or  are  the  receptors  in  whom 
this  function  resides  other  than  the  normal  antibodies  which  are 
present  in  the  blood;  should  we,  in  other  words,  seek  for  them  in 
the  protoplasm  of  the  nerve  cell,  for  instance,  when  dealing  with 
diphtheria  toxin?  Ehrlich's  theory,  to  be  sure,  harmonizes  with 
either  alternative,  but  it  would  scarcely  seem  that  it  is  ever  indica- 
ted which  of  the  two  should  be  accepted. 

In  certain  other  respects  Ehrlich's  theory  has  apparently  been 
more  precise.  It  attempted,  at  a  stage  when  the  data  were  still  very 
limited,  to  interpret  the  mode  of  action  of  antibodies  with  antigens, 
here  it  seems  to  me  that  it  has  exercised  a  perturbing  influence  on 


A  GENERAL  RESUME   OF  IMMUNITY.  499 

the  progress  of  knowledge,  and  has  really  hindered  the  free  de- 
velopment of  investigation.  In  offering  explanations  which  seem 
definitive,  and  schemata  which  satisfy  the  experimenter  and 
appease  his  curiosity,  Ehrlich's  theory  has  come  to  make  certain 
problems,  which  have  scarcely  been  touched  upon,  regarded  as 
worked  out.  It  must  be  admitted  that  its  partisans  have  seemed 
chiefly  preoccupied  with  justifying  it,  and  it  would  seem  as  if  their 
efforts  were  directed  rather  toward  defending  the  theory  than  con- 
trolling it.  And  the  guiding  thought,  to  my  thinking  fatal,  which 
they  have  endeavored  to  enforce,  is  the  constant  attribution  to  the 
molecule  of  the  antibody  of  separate  atom  groups  for  each  of  the 
phenomena  to  which  the  antibody  gives  rise.  To  each  manifesta- 
tion that  has  been  observed  there  must  be  a  corresponding  molec- 
ular group  or  even  a  new  molecule.  Such  a  method  is  favorable  to 
the  theory,  inasmuch  as  each  influence  as  noted  finds  material 
evidence  in  a  special  group  which  becomes  a  sort  of  sub-stratum  for 
it.  Its  use,  however,  is  doubtful  for  the  same  reason,  for  it  gives  too 
easily  the  appearance  of  an  explanation,  when  in  reality  it  estab- 
lishes no  definite  relation  between  the  phenomena  that  have  been 
observed,  since  it  satisfies  itself  in  symbolizing  these  manifestations 
by  independent  groups,  each  of  which  appears  as  a  resting  place  for 
such  and  such  a  property.  These  groups  are  simply  evoked  by  the 
theorist  as  he  wishes  and  their  very  existence  in  each  and  every  case 
is  far  from  sure.  When  we  say  that  an  agglutinin  which  unites  with 
bacteria  and  clumps  them  produces  these  results  because  it  possesses 
two  groups  in  its  molecule,  one  of  which  combines  and  the  other  of 
which  agglutinates,  it  facilitates  explanation,  and  seems,  indeed, 
and  in  this  its  danger  lies,  to  resolve  the  question  entirely,  but  it 
is  probably  inexact.  In  the  first  place,  it  is  not,  strictly  speaking, 
the  agglutinin  which  agglutinates,  but  rather,  as  I  showed  in  1899, 
the  salt.  There  are  antigens  which,  when  united  with  their  anti- 
bodies, form  complexes  that  have  the  characteristic  of  being 
flocculable  by  electrolytes.*  It  is  the  complex  which  agglutinates 
and  there  is  no  reason  to  localize  the  cause  of  agglutination  in  a 

*  In  this  respect  there  are  analogies  between  bacteria  laden  with  agglutinin 
and  colloidal  complexes,  not  only  in  respect  to  the  action  of  salts  but  also  to  the 
influence  of  electricity.  According  to  Neisser  and  Friedemann,  the  complex  mas- 
tic-gelatin is  flocculable  by  the  electric  current  just  as  bacteria  that  have  fixed 
agglutinin  are. 


500  STUDIES  IN  IMMUNITY. 

molecule  of  the  antibody  rather  than  in  one  of  the  antigen.  The 
hypothesis  of  a  functional  group  in  the  molecule  of  the  agglutinin 
is  all  the  more  doubtful  inasmuch  as  it  is  not  the  only  substance 
which  can  render  bacteria  sensitive  to  the  flocculating  action  of 
salts.  Bacteria  that  have  absorbed  iron,  uranium,  or  aluminium 
compounds  are  subsequently  flocculableby  salts  (Neisser  and  Friede- 
mann,  Bechhold,  Gengou) ;  silicic  acid  is  similar  in  its  action  (Land- 
steiner  and  Jagic). 

It  has  been  found  that  agglutinins  when  heated  may  keep  the 
property  of  uniting  with  bacteria  although  they  lose  the  property 
of  agglutinating  them  (Michaelis,  Eisenberg  and  Volk,  Bail).  And 
to  explain  this  fact  it  has  been  said  that  under  these  conditions  the 
agglutinin  loses  its  agglutinating  group  but  keeps  its  combining 
group.*  Certain  bacteria  act  in  the  same  way  as  agglutinins.  The 
typhoid  bacillus,  when  heated  to  80  degrees,  still  fixes  agglutinin  but 
is  not  agglutinable  (Weil).t  An  aqueous  solution  of  agar,  so  diluted 
as  to  be  only  slightly  viscuous  at  room  temperature,  agglutinates 
barium  sulphate  suspended  in  water.  Heating  such  a  solution 
destroys  this  property  without  affecting  the  adsorbing  property: 
under  these  conditions  it  produces  the  opposite  effect,  namely,  dis- 
seminates the  particles  of  barium  and  gives  a  milky  appearance  to 
the  fluid  (Gengou).  Can  we  say  here  that  by  heating  this  solution 
we  have  caused  it  to  lose  its  agglutinating  molecular  group?  As 
Forges  has  already  stated,  the  hypothesis  of  the  existence  of  such  a 
group  in  the  antibody  molecule,  has  no  foundation;  he  found  on 
studying  the  effect  of  heat  on  the  agglutinating  power  of  the  albu- 
minous substances  of  serum  for  mastic  emulsions,  that  he  could 
obtain  entirely  similar  results  to  those  that  have  been  noted  for 
agglutinins  and  bacteria.  {  Any  change  in  the  physical  properties 
of  the  constituents  of  the  complex,  agglutinin-bacterium,  and  par- 
ticularly as  regards  the  degree  of  colloidal  stability,  may  influence 
the  state  of  equilibrium  of  this  complex  in  respect  to  the  surround- 
ing fluid,  and  on  this  state  of  equilibrium  the  phenomenon  of  agglu- 

*  It  is  well  known  that  precipitins  give  similar  results  (Kraus  and  von  Pirquet) . 

f  This  same  author  has  shown  that  B.  typhosus,  which  under  normal  condi- 
tions isfeebly  agglutinated  by  gelatin,  loses  this  property  when  heated  to  SOdegrees. 

t  Neisser  and  Friedemann  and  Bechhold  have  contributed  interesting  results 
on  the  effect  of  gelatin,  serum  and  the  like,  on  the  agglutination  of  mastic  by 
electrolytes. 


A  GENERAL  RESUME  OF  IMMUNITY.  501 

tination  depends.  In  short,  the  essential  phenomenon  with  agglu- 
tinins, as  with  other  active  substances  in  sera,  is  its  union  with  the 
antigen;  as  far  as  the  agglutination  itself,  which  follows  this  union, 
is  concerned,  it  is  only  a  secondary  phenomenon  on  which  we  can- 
not depend  in  considering  agglutinins  as  functionally  different 
in  molecular  structure  from  the  other  antibodies.  Such  remarks  on 
agglutinins  also  apply  to  precipitins  or  the  sensitizers  which  Ehrlich 
endows  with  a  complementophilic  group.  It  is  particularly  be- 
cause this  investigator's  theory  suggested  an  artificial  classification 
of  the  antibodies,  that  it  appears  to  me  to  have  exerted  a  harmful 
influence  on  scientific  progress. 

Everyone  agrees,  naturally,  that  the  numerous  antibodies  which 
the  study  of  immunity  has  brought  to  our  knowledge  and  which 
are  active  on  such  different  elements  as  bacteria,  cells,  toxins,  and 
the  like,  should  not  be  considered  as  identical,  inasmuch  as  they  may 
be  distinguished  as  regards  specificity,  or,  in  other  words,  since  they 
unite  with  different  antigens.  But  in  addition  to  this  incontestable 
difference  of  specificity,  Ehrlich  has  imagined  another  one  which  is 
more  far  reaching  and  which  deals  with  the  molecular  structure 
of  the  antibody.  Indeed,  he  classes  these  antibodies  in  ac- 
cordance with  their  molecular  structure  into  three  genera;  anti- 
toxins with  a  single  combining  group;  agglutinins  and  precipitins 
with  a  single  combining  group  but  with  an  additional  functional 
group  which  brings  about  agglutination  or  precipitation ;  and  finally 
sensitizers  which  have  two  combining  groups  in  their  molecule, 
uniting  on  the  one  hand  with  the  cell  that  is  affected  and  on  the 
other  with  the  alexin  (complement),  and  hence  the  name  of  ambo- 
ceptor. 

In  every  instance  according  to  this  classification  the  phenomena 
observed  are  attributed  to  special  properties  in  the  antibody  and 
never  to  those  in  the  antigen.  As  a  matter  of  fact,  these  phenomena 
should  be  related,  not  as  regards  antigen  or  antibody  considered 
separately,  but  as  regards  the  complexes  which  result  from  their 
union,  and  it  is  evident  that  the  special  properties  of  the  antigen 
must  affect  markedly  and  perhaps  to  a  preponderating  degree,  the 
qualities  of  such  complexes.  I  have  just  mentioned  this  idea  as 
applied  to  agglutination,  and  I  have  frequently  defended  it  in 
respect  to  sensitizers.  The  sensitizer,  on  uniting  with  the  cell 


502  STUDIES   IN  IMMUNITY. 

which  it  affects,  forms  with  it  a  complex  endowed  with  avidity  for 
alexin,  a  complex  which,  in  other  terms,  manifests  properties  of 
adsorption  which  neither  of  its  constituents  alone  possesses.  Just 
as  the  union  of  agglutinins  with  bacteria  produces  in  them  a  remark- 
able sensitivity  to  the  agglutinating  effect  of  electrolytes  by  modi- 
fying their  property  of  molecular  adhesion,  in  a  similar  way  sensi- 
tizers  confer  on  their  antigens  a  similar  modified  property  of  adhe- 
sion, namely,  alexin  adsorption.  Such,  indeed,  is  my  conception 
of  sensitization,  and  I  shall  later  return  briefly  to  a  discussion  of  it 
in  considering  objections  that  have  been  raised  to  it. 

Nothing  authorizes  us  to  separate  the  antibodies  with  which  we 
have  been  dealing  by  such  sharp  damarcations  as  to  attribute  to 
them  such  different  constitutions  or  as  to  suppose  that  the  cause  of 
all  these  observed  manifestations  is  due  to  changes  in  their  mole- 
cule, without  paying  any  attention  to  the  nature  of  the  antigen. 
Antibodies  of  widely  divergent  appearance  present,  then,  marked 
relationship  to  one  another,  and  the  separations  that  have  been 
raised  by  classifying  them  in  accordance  with  hypotheses  dealing 
with  the  general  structure  of  their  molecule,  are  imaginary.  In  my 
opinion,  antibodies,  whatever  their  nature,  act  very  much  alike; 
but  the  effects  that  they  produce  differ  with  the  antigen  in  question 
and  the  characteristics  which,  on  account  of  its  own  nature,  it  can 
produce  as  soon  as  it  unites  with  the  appropriate  antibody.  It  is 
evident  that  this  point  of  view,  in  addition  to  being  better  in  har- 
mony with  fact,  presents  also  the  marked  advantage  of  producing 
a  greater  unity  in  our  conception  of  the  properties  of  sera,  since  it 
does  not  necessarily  recognize  various  families  of  antibodies,  but 
limits  itself  to  noting  the  infinite  variety  of  the  antigens. 

*  ** 

It  is  not  for  me  to  defend  my  work,  which  is  here  offered  in  its 
entirety  to  the  judgment  of  the  scientific  world.  But  I  may  be  per- 
mitted to  characterize  my  method  of  research  by  saying  that  I  have 
yielded  as  little  as  possible  to  the  inspiration  of  theory;  and  for  this 
reason,  moreover,  no  general  conception  of  obscure  questions  will 
be  found  in  the  present  article.  Like  every  other  observer,  I  have 
offered  certain  hypotheses,  but  they  scarcely  merit  this  name,  for 
they  are  so  little  removed  from  the  facts  observed;  they  are  rather 
a  transcription  of  impressions  gathered  from  the  results  of  labora- 


A  GENERAL  RESUME   OF   IMMUNITY.  503 

tory  experimentation.  At  the  risk  of  being  considered  by  some 
readers  as  not  possessing  a  sufficiently  generalizing  mind,  I  must 
admit  that  I  have  been  led  to  make  my  most  important  discoveries 
by  yielding  tractably  to  the  impulse  of  facts,  by  letting  myself  be 
moved  by  my  data  without  attempting  to  discipline  them  or  sub- 
ject them  to  systematic  ideas  of  my  own.  This  is  quite  evident  to 
one  who  follows  the  evolution  of  my  researches.  For  example,  I 
have  frequently  been  asked  how  I  came  to  discover  the  law  of  bac- 
teriolysis (that  is  to  say  the  collaboration  of  two  substances,  alexin 
and  sensitizer),  or  how,  following  that  deduction,  I  had  the  idea  of 
immunizing  animals  against  the  blood  of  an  alien  species  or  against 
milk  (hemolytic  sera  and  precipitins  for  albuminous  substances), 
which  enabled  me  to  demonstrate  the  idea  that  the  organism,  by 
employing  the  same  mechanism  and  by  the  same  proceedings, 
immunizes  itself  against  elements  that  differ  markedly  from  bac- 
teria; that  is  to  say,  the  production  of  antibacterial  antibodies 
simply  represents  the  application,  in  the  struggle  against  infective 
agents,  of  a  faculty  which  the  animal  would  have  possessed  even  if 
contagious  disease  had  not  existed.  I  may  be  permitted  then  to 
note  how  these  researches  have  been  related  to  one  another,  and  it 
will  be  evident  that  no  guiding  theory  has  been  necessary. 

Pfeiffer  had  just  discovered  lysis  of  the  cholera  vibrio  in  the  peri- 
toneum of  guinea-pigs,  and  Metchnikoff  had  shown  that,  contrary 
to  Pfeiffer's  opinion,  this  phenomenon  occurs  in  vitro  as  well  as  in 
the  living  animal;  all  that  was  necessary  was  to  mix  a  little  of  the 
peritoneal  exudate  from  a  normal  guinea-pig  with  cholera  vibrios 
to  which  a  trace  of  cholera  serum  had  been  added.  I  wondered 
whether  the  exudate  could  be  replaced  by  fresh  defibrinated  blood 
from  a  normal  animal.  I  found  that  it  could ;  vibrios  mixed  with 
it  in  the  presence  of  cholera  serum  showed  granular  transformation. 
The  question  then  was  whether  it  was  the  cells  or  the  serum  in  the 
defibrinated  blood  which  produce  this  effect,  and  experimentally  I 
found  that  the  serum  was  the  important  substance  and  that  neither 
red  nor  white  corpuscles  were  necessary.  I  could  definitely  dis- 
card the  idea  of  cellular  participation.  But  why  was  fresh  normal 
serum  necessary?  The  cholera  serum,  as  we  then  employed  it  in 
experiments,  usually  came  from  a  stock  that  had  been  kept  for  some 
time,  or,  as  frequently  happened,  had  been  heated  to  60  degrees. 


504  STUDIES  IN  IMMUNITY. 

It  was  quite  reasonable  to  presume  that  freshly  obtained,  unheated 
cholera  serum  should  act  both  as  an  immune  serum  and  as  fresh 
normal  serum;  and  this,  indeed,  was  what  happened.  Under  these 
conditions  it  alone  produced  metamorphosis  of  the  cholera  vibrio. 
When  heated  to  55  degrees  it  lost  its  bacteriolytic  power,  but  recov- 
ered it  on  the  addition  of  fresh  normal  serum.  Such  was  the  dem- 
onstration of  the  two  substances,  the  collaboration  of  which  is 
necessary  to  produce  bacteriolysis, — the  sensitizer  or  preventive  sub- 
stance, thermostable,  specific,  and  characteristic  of  immune  serum, 
and  the  alexin  destroyed  at  55  degrees,  — non-specific,  and  present  in 
practically  the  same  amount  in  the  sera  of  normal  and  of  vaccinated 
animals,  as  may  be  shown  by  proper  mensuration.  While  carrying 
out  these  experiments  (1895),  it  was  noted  that  cholera  serum  im- 
mobilizes and  agglutinates  cholera  vibrios  in  clumps;  this  was  the 
first  instance  of  agglutination  of  a  bacterial  culture  on  the  addition 
of  a  small  dose  of  immune  serum.  It  was  further  found  that  this 
agglutination  would  still  occur,  even  with  cholera  serum  that  had 
been  heated  to  60  or  65  degrees,  from  which  fact  it  was  evident  that 
the  agglutinating  property  is  separate  from  the  bactericidal  property. 
It  became  obvious  at  once  that  one  might  use  this  test  which  is  so 
easy  to  perform  in  vitro,  in  the  diagnosis  of  the  cholera  vibrio, 
instead  of  the  in  vivo  method  which  Pfeiffer  discovered.  This 
marked  the  introduction  in  bacteriology  of  serum  diagnosis  in  vitro. 

As  may  be  seen,  the  idea  of  a  participation  of  two  substances  in 
bacteriolysis  is  not,  properly  speaking,  a  theory,  for  there  is  nothing 
hypothetical  about  it;  it  is  simply  a  literal  translation  of  observed 
facts,  particularly  the  experiments  of  " reactivation"  which  have 
just  been  mentioned.  Following  is  an  account  of  the  discovery  of 
hemolytic  sera: 

In  my  experiments  in  bacteriolysis  I  frequently  used  as  an  im- 
mune serum,  the  serum  of  a  goat  that  had  been  immunized  against 
the  cholera  vibrio,  and  as  alexin  (complement),  fresh  normal  guinea- 
pig  serum.  It  frequently  happened  that  the  latter  contained  a  cer- 
tain number  of  red  blood  cells  and  I  found  that  these  were  agglu- 
tinated and  would  be  agglutinated  even  when  mixed  with  normal 
goat  serum.  I  had,  moreover,  already  noted  that  neither  motility 
nor  vitality  was  a  necessary  condition  for  the  agglutination  of  bac- 
teria; cholera  serum  agglutinates  vibrios  that  have  been  killed  by 


A  GENERAL  RESUME   OF  IMMUNITY.  505 

chloroform  very  well.  Since  the  bacteria  then  are  passive  agents, 
red  blood  cells  may  be  similarly  considered.  I  had  further  noted 
(and  this  fact  was  likewise  mentioned  at  about  the  same  time  by 
Gruber  and  Durham),  that  normal  sera  (and  particularly  certain  of 
them  such  as  horse  serum),  frequently  show  an  agglutinating  prop- 
erty for  various  bacteria  (for  example,  V.  cholerae,  B.  typhosus, 
B.  tetani).  This  constitutes  a  second  relation  between  bacteria  and 
red  blood  cells,  inasmuch  as  the  latter  are  also  frequently  aggluti- 
nated by  normal  sera  of  alien  species.  It  then  occurred  to  me, 
naturally  enough,  that  if  immunization  against  bacteria  increases 
the  agglutinating  property  toward  a  given  organism  to  a  consider- 
able extent  over  that  in  the  normal  animal,  we  might  hope  to  obtain 
a  similar  result  with  red  blood  cells.  By  immunizing  an  animal  of 
species  A  with  the  blood  of  species  B,  we  should  obtain  in  animal  A, 
a  powerful  agglutinin  for  the  corpuscles  of  B.  With  this  purpose  in 
mind  I  vaccinated  guinea-pigs  against  rabbit  blood.  When  I  saw 
that  specific  hemolysis  also  occurred  under  these  conditions,  the 
analogy  of  which  with  bacteriolysis  was  most  obvious,  I  had  simply 
to  repeat  the  experiments  that  I  had  already  performed  with  cholera 
serum  and  cholera  vibrios  with  hemolytic  sera  and  corpuscles.  The 
law  of  two  substances  was  completely  verified,  and  its  significance 
and  importance  became  more  evident. 

My  researches  on  "  lac  to  serum,"  that  is  to  say  an  immune  serum 
which  precipitates  milk,  as  well  as  the  ideas  which  I  obtained  con- 
cerning, first,  the  mechanism  of  agglutination,  and  later,  the  nature 
of  the  reactions  between  antibodies  and  antigens,  which,  according 
to  my  idea,  are  probably  to  be  classed  in  a  category  of  adsorption 
phenomena,  also  began  quite  independently  of  any  abstract  concep- 
tion. On  examining  test  tubes  which  contained  bacteria  aggluti- 
nated by  sera,  I  had  been  struck  with  the  resemblance  between 
their  appearance  and  the  flocking  out  of  chemical  precipitates,  par- 
ticularly of  the  insoluble  salts  coming  from  a  double  decomposition. 
It  is  a  current  notion  that  flocculation  is  favored  by  electrolytes,  and 
the  influence  of  the  latter  had  been  particularly  studied  in  respect  to 
the  sedimentation  of  clay  and  the  agglutination  of  emulsions  of  mas- 
tic. A  study  as  to  whether  the  agglutination  of  bacteria  also  neces- 
sitated the  presence  of  salts  was  obvious,  and  as  we  know,  such  a 
necessity  was  experimentally  proved.  It  was  found  that  bacteria 


506  STUDIES  IN  IMMUNITY. 

which  had  been  affected  by  the  agglutinin,  act  subsequently  pre- 
cisely like  inert  chemical  particles.  In  other  words,  bacteria,  on 
uniting  with  the  agglutinin,  give  a  coagulum  which  is  flocculable  by 
electrolytes.  But  could  we  not  go  further  and  say  that  any  easily 
coagulable  substance  will  give  the  same  results?  Should  we  not 
obtain  an  agglutinating  serum  following  the  injection,  not  of  formed 
elements  like  bacteria,  but  of  particles  of  amorphous  organic  sub- 
stances? Milk  casein,  which  occurs  in  a  colloidal  condition  in  the 
form  of  extremely  minute  particles,  is  suitable  to  prove  this  point. 
Animals  that  were  immunized  against  milk  gave  a  serum  which 
precipitated  casein  and  consequently  agreed  with  the  observations 
which  Tchisto witch  had  just  made  on  the  formation  of  a  precipitate 
on  adding  eel  serum  to  its  antitoxin. 

The  flocculation  of  milk  casein  by  the  corresponding  immune 
serum  established  a  new  connection  between  the  phenomena  of 
agglutination  and  coagulation  which  Duclaux  had  previously 
asserted  to  be  closely  related.  Adsorption  of  the  agglutinin  modi- 
fies bacteria  in  respect  to  their  properties  of  adhesion  with  the 
surrounding  fluid,  and  this  modification  is  evident  by  their  sus- 
ceptibility to  electrolytes.  But  how  does  the  agglutinin  unite?  Wo 
might  conceive  of  a  single  molecular  contact,  of  an  adsorption  of  the 
antibody  by  the  antigen  similar  to  the  adsorption  of  anilin  dye  by 
filter  paper.  I  tested  antibodies  from  this  standpoint,  and  in  par- 
ticular did  the  experiment  which  demonstrated  that  a  given  dose  of 
hemolytic  serum  destroys  variable  amounts  of  corpuscles  in  accord- 
ance with  their  addition  in  a  single  or  in  several  doses.  It  would 
take  me  too  far  afield  to  discuss  this  experiment  in  greater  detail, 
an  experiment  which,  as  we  know,  has  been  employed  by  various 
investigators  with  similar  results,  particularly  in  dealing  with 
antitoxins.  Such  are  the  purely  experimental  results  which  first 
suggested  to  me  the  ideas  that  I  have  since  defended  concerning 
the  mode  of  action  of  antibodies,  without  being  guided  by  any 
theoretical  leaning  in  one  direction  more  than  another. 

When  we  have  hemolytic  sera  which  are  evidently  toxic,  it  is 
quite  natural  to  attempt  to  obtain  antitoxins  to  them;  the  effect  of 
such  an  antiserum  on  the  hemolytic  serum  can  then  be  split  up  into 
several  factors  and  it  is  found  to  contain  both  an  antisensitizer  and 
an  anti-alexin.  From  another  standpoint,  inasmuch  as  the  alexin 


A  GENERAL  RESUME  OF  IMMUNITY.  507 

either  hemolyzes  or  bacteriolyzes,  it  is  necessary  to  determine 
whether  it  is  used  up  in  its  action,  and  whether  the  surrounding 
fluid  becomes  inactive  for  new  sensitized  cells,  whether  corpuscles  or 
bacteria.  By  such  experiments  the  fixation  of  the  alexin,  and  at  the 
same  time  its  functional  unity,  were  determined  —  but  here  let  me 
stop  in  this  review  of  the  evolution  of  my  researches.  It  is  quite 
useless  to  go  further,  as  my  single  desire  has  been  to  show  that  it  is 
not  I,  myself,  who  has  created  or  even  chosen  my  ideas;  they  have 
rather  been  forced  upon  me  by  facts,  by  the  logical  induction  which, 
so  to  speak,  is  the  inevitable  result  of  experimentation,  and  by  the 
immediate  deductions  from  it.  I  have  limited  myself  to  being  an 
experimenter  and  very  little  of  a  theorist,  and  the  accuracy  of  the 
opinions  which  I  have  defended  is,  I  think,  best  guaranteed  by  this 
fact.  Certain  of  my  conclusions  which  have  been  attacked  for  a 
long  time  are  now  beginning  to  gain  the  approbation  of  investiga- 
tors. Such,  for  example,  is  the  idea  of  a  functional  unity  of  alexin 
(complement),  and  the  idea  that  sensitizers  possess  no  complemento- 
philic  group  but  form  a  complex  with  the  antigen  which  manifests 
adsorption  properties  for  alexin.  It  would  seem  well,  perhaps,  to 
consider  briefly  the  present  status  of  these  two  subjects  of  discus- 
sion, and  to  sum  up  the  arguments  for  and  against  them. 

*** 

Alexins  .derived  from  animals  of  different  species  are  not  all  en- 
dowed with  the  same  properties;  they  are  particularly  to  be  dis- 
tinguished in  that  they  are  not  all  equally  toxic  and  equally  apt  to 
produce  hemolysis  and  bacteriolysis.  I  do  not  need  to  insist  further 
on  this  point,  inasmuch  as  it  has  been  accepted  by  nearly  all  ob- 
servers. The  researches  of  Streng,  which  were  done  at  our  Pasteur 
Institute,  demonstrated  the  real  existence  of  anti-alexins  which  had 
been  contested  by  certain  authors,  and  still  further  showed  the  vary- 
ing toxicity  of  different  alexins. 

But  is  there  a  single  alexin  in  a  given  serum?  Without  citing  in 
detail  all  the  arguments  in  favor  of  it  which  have  been  offered  in 
my  articles,  I  wish  again  to  insist  that  this  problem  should  be  con- 
sidered exclusively  from  the  standpoint  of  function.  Our  knowl- 
edge of  the  constitution  of  the  alexin  is  too  obscure  to  permit 
our  determining  whether  the  alexin  in  a  given  serum  is  chemi- 
cally one  or  more  substances.  The  important  point  to  know  is 


508  STUDIES  IN  IMMUNITY. 

whether  a  given  alexin  reacts  with  various  cells  such  as  bacteria  and 
corpuscles.  In  1895  I  thought  to  outline  the  process  of  immuniza- 
tion as  follows: 

"The  bactericidal  substance  (alexin)  is  the  same  whatever  be 
the  bacterium  in  question.  In  animals  vaccinated  against  certain 
infections  the  energy  of  the  bactericidal  substance  is  more  markedly 
evident  against  a  particular  bacterium,  owing  to  the  effect  of  the 
preventive  substance  (sensitizer)  which  varies  in  each  case  and  the 
nature  of  which  depends  on  the  micro-organism  used  for  immuniza- 
tion. It  is  owing  to  the  intervention  of  this  particular  substance, 
the  specific  antibody,  that  the  animal  body  directs  its  destructive 
power  against  a  particular  infective  agent." 

Is  this  statement  still  accurate  in  spite  of  the  affirmations  to  the 
contrary  as  regards  a  multiplicity  of  alexins  which  have  come  from 
Ehrlich  in  particular?  At  that  time  I  expressed  in  a  definite  man- 
ner the  opinion  which  was  later  confirmed  by  a  study  of  hemolytic 
sera,  that  the  animal  body  does  not  contain  a  series  of  alexins, 
some  of  which  act  in  the  presence  of  certain  sensitizers  and  are  thus 
employed  in  struggling  against  certain  bacteria,  while  others  are 
better  adapted  to  work  with  other  sensitizers  to  combat  other  bac- 
teria. On  the  contrary  it  is  the  same  weapon  in  each  instance, 
a  single  alexin  which  reacts  now  against  one  and  now  against 
another  bacterium,  owing  to  the  specificity  of  the  sensitizer.  Sev- 
eral of  my  articles,  particularly  the  one  on  cytolytic  sera  in  1901, 
gave  the  experimental  foundation  for  this  conception,  and  I  may 
content  myself  simply  with  recalling  them. 

If  this  conception  were  incorrect,  if,  for  example,  the  hemolytic 
alexin  differed  from  the  bacteriolytic  alexin,  how  would  Gengou  and 
I  have  been  able  to  devise  an  invariable  method  for  demonstrating 
anti-bacterial  sensitizers,  namely,  the  fixation  of  the  alexin  by  sensi- 
tized bacteria,  as  shown  by  using  sensitized  red  blood  cells  as  a 
reagent?  Would  this  method  have  been  universally  accepted  as  it 
has  been?  Should  we  have  been  able  to  use  it  as  an  argument  in 
favor  of  the  authenticity  of  our  organism  of  whooping  cough?  If  the 
conception,  which  is  the  basis  of  this  method,  were  false,  what  con- 
fidence would  remain  in  the  serum  diagnosis  of  syphilis?  Notwith- 
standing, the  contrary  thesis  of  a  multiplicity  of  alexins  in  a  given 
serum  met  with  the  support  of  many  investigators  a  few  years  ago. 


A  GENERAL  RESUME  OF  IMMUNITY.  509 

I  believe  this  is  owing  to  the  fact  that  in  interpreting  experiments 
insufficient  attention  was  paid  to  two  essential  points,  the  first  of 
which  is  the  antagonistic  power  of  serum,  and  the  other  that  a  given 
alexin  does  not  destroy  all  cells  with  equal  facility.  Gay  and  I  have 
given  over  an  entire  article  to  the  study  of  the  antagonistic  property 
of  serum,  and  have  called  attention  to  the  fact  (which  was  likewise 
brought  out  by  the  researches  of  Muir  and  Browning  in  the  same 
direction)  that  blood  corpuscles,  particularly  when  feebly  sensi- 
tized, may  remove  only  a  small  amount  of  the  alexin  from  a  sur- 
rounding fluid,  even  when  they  are  present  in  large  numbers.  The 
same  idea  applies,  of  course,  to  bacteria.  Such  a  fact  destroys 
all  the  value  of  the  so-called  "elective  complement  absorption 
method."  The  fact  that  in  the  presence  of  a  sensitized  cell  a  cer- 
tain portion  of  the  alexin  may  remain  free  in  the  surrounding  fluid 
does  not  in  any  way  prove  that  this  non-absorbed  alexin  differs  from 
that  which  has  been  fixed.  This  remark  also  applies  to  experiments 
in  which  weakly  sensitizing  sera  are  used  (normal  sera  for  example), 
experiments  that  have  been  frequently  used  to  demonstrate  a  func- 
tional multiplicity  of  the  alexin. 

In  the  second  place,  when  we  note  that  a  small  dose  of  a  given 
alexic  serum  suffices  to  destroy  sensitised  corpuscles  of  species  A, 
whereas  a  larger  amount  of  the  same  serum  is  necessary  to  destroy 
sensitized  corpuscles  B,  we  are  not  authorized  to  conclude  that  two 
different  alexins  are  present,  each  one  appropriate  for  only  one 
of  these  two  species  of  corpuscles,  and  that  one  of  these  alexins 
exists  in  larger  amount  than  the  other.  In  accordance  with  the 
work  of  Muir  and  Browning,  and  of  Gay  especially,  we  must  con- 
clude that  there  is  only  one  alexin,  but  that  corpuscles  differ  in  the 
amount  of  this  substance  which  they  require  for  hemolysis. 

It  is  further  to  be  noted  that  the  partisans  of  a  functional  multi- 
plicity of  the  alexin  have  since  modified  their  original  conception  to 
a  great  extent.  They  now  admit  that  various  amboceptors  from  a 
given  animal  species  have  a  uniform  structure  in  so  far  as  the  com- 
plementophilic  group  is  concerned,  which  is  equivalent  to  saying  that 
they  absorb  the  same  alexin.  If  we  are  to  admit,  then,  that  various 
hemolytic  or  bacteriolytic  sensitizers  produce  fixation  of  the  same 
alexin  on  various  cells  which  they  affect,  we  evidently  admit  that 
these  alexins  may  be  used  indifferently  in  hemolysis  and  bacterioly- 


510  STUDIES  IN  IMMUNITY. 

sis,  in  other  words,  we  admit  the  unity  of  the  alexin  at  least  func- 
tionally, if  not  chemically.  In  conclusion,  then,  I  think  that  the 
opinion  which  I  expressed  in  1895  on  the  mechanism  of  acquired 
immunity  in  accordance  with  which  the  animal  body  would  seem  to 
direct  the  same  weapon,  the  same  alexin  against  various  foreign 
cells,  owing  to  the  presence  of  a  specific  sensitizer,  is  gradually 
receiving  the  united  agreement  of  scientists. 

In  so  far  as  concerns  the  mode  of  fixation  of  alexin,  the  divergent 
opinions  of  Ehrlich  and  Morgenroth,  and  myself,  are  well  known. 
According  to  these  authors,  the  sensitizers  have  a  complemento- 
philic  group.  I  have  already  previously  mentioned  my  own 
opinion;  I  think  that  avidity  for  alexin  logically  resembles  agglu- 
tination or  sensitivity  to  the  flocculating  action  of  salts.  Taken 
alone,  neither  the  bacteria,  or  at  least  the  great  majority  of  them, 
nor  the  agglutinin  is  affected  by  salt,  but  the  complex  which  they 
form  is  agglutinable  by  this  electrolyte.  In  the  same  way  neither 
the  amboceptor  nor  the  antigen  in  question  has  alone  any  manifest 
affinity  for  the  alexin,  but  they  form  by  their  union  a  complex 
which  can  absorb  alexin,  in  other  words,  which  has  particular 
properties  of  adhesion.  As  a  result,  as  I  have  already  noted,  there 
is  no  fundamental  difference  between  sensitizers  and  such  other 
antibodies  as  antitoxins.  There  is  no  such  thing  as  an  ambocep- 
tor; all  antibodies  on  the  contrary  are  "uniceptors."  There  exist, 
however,  antigens  which,  by  uniting  with  the  suitable  antibody, 
give  complexes  which  can  adsorb  alexin,  whereas  other  antigens 
have  no  such  property.  I  do  not  think  it  is  necessary  for  me  to 
return  to  the  experiments  that  I  have  described  in  my  articles  which 
show  that  there  is  no  means  of  determining  any  direct  affinity  of 
sensitizers  for  alexin  in  absence  of  the  antigen,  and  consequently 
there  is  no  reason  for  admitting  the  existence  of  a  complemento- 
philic  group.*  Other  experimenters  have  brought  out  similar  facts 
which  lead  to  the  same  conclusions.  Thus,  according  to  Frouin  and 
Muir  and  Browning,  on  passing  serum  through  certain  filters  it  is 
found  that  the  sensitizers  pass  through  whereas  the  alexin  is  retained. 

*  This  is  true,  not  only  as  regards  sensitizers  that  act  on  corpuscles  or  bacteria, 
but  also,  as  Gengou  has  shown,  as  regards  those  that  affect  albuminoid  substances; 
in  this  case  also  fixation  of  alexin  takes  place  only  when  both  the  antibody  and 
the  antigen  are  present. 


A  GENERAL  RESUME  OF  IMMUNITY.  511 

In  a  similar  manner  these  same  English  authors  have  recently  found 
that  sensitizers  filter  equally  well  whether  mixed  or  not  with  a 
strong  dose  of  complement,  which  latter  substance,  as  already  men- 
tioned, does  not  succeed  in  getting  through.  Ehrlich  and  Morgen- 
roth,  to  be  sure,  had  already  made  similar  observations  which 
corroborate  this  idea  that  the  alexin  and  the  sensitizer  exist  side 
by  side  in  serum  without  being  combined.  I  established  that  par- 
ticipation of  the  antigen  is  always  necessary  in  alexin  absorption. 
Partisans  of  the  complementophilic  group  now  admit  that  such  a 
conception  is  true  in  at  least  the  majority  of  cases;  they  say,  in 
fact,  that  the  energy  of  affinity  of  the  complementophilic  group 
is  frequently  increased  as  a  result  of  a  union  of  the  sensitizer 
with  the  antigen.  This  amounts  essentially  to  accepting  the  no- 
tion, to  my  opinion  the  only  important  one,  that  fixation  of  alexin 
is  subsequent  to  the  formation  of  the  complex,  antibody-antigen. 
And  what  is  more,  the  hypothesis  of  a  complementophilic  group  is 
in  distinct  disagreement  with  certain  of  Muir's  experiments.  This 
author  found  that  blood  corpuscles  that  had  fixed  the  sensitizer  and 
had  been  saturated  with  alexin  could  subsequently,  by  diffusion, 
lose  a  certain  amount  of  their  sensitizer,  although  they  retain  the 
complement,  and  what  is.  more,  in  this  instance  they  lose  as  much 
sensitizer  as  if  they  had  not  absorbed  complement.  Consequently 
it  is  in  no  way  through  the  mediation  of  the  sensitizer  that  the  alexin 
attaches  itself  to  the  corpuscles;  if  this  were  the  case  the  removal 
of  the  sensitizer  would  necessarily  imply  that  of  the  alexin,  which, 
as  we  have  just  mentioned,  does  not  leave  the  corpuscles. 

It  is  clear  that  if  it  had  been  proved  that  the  sensitizer  fixes 
the  alexin  without  the  presence  of  the  antigen  in  even  a  single 
instance,  the  idea  of  a  complementophilic  group  should  be  accepted. 
Ehrlich  and  Sachs,  as  we  know,  thought  that  they  had  found  this 
decisive  example  in  dealing  with  the  hemolysis  of  guinea-pig  cor- 
puscles by  a  mixture  of  heated  bovine  serum  and  fresh  horse  serum. 
But  the  researches  I  carried  out  with  Gay  are  also  known,  researches 
which  I  have  subsequently  continued  and  in  accordance  with  which 
Ehrlich  and  Sachs'  interpretation  is  completely  inaccurate.  These 
researches  have  been  criticised  by  Sachs  and  Bauer,  which  accounts 
for  my  reconsideration  of  the  subject  in  collaboration  with  Dr. 
Oswald  Streng.  Streng  and  I  were  able  to  prove  that  Sachs  and 


512  STUDIES  IN  IMMUNITY. 

Bauer's  objection  is  without  foundation,  and  we  have  denned  still 
further  the  properties  of  that  remarkable  substance  which  we  for- 
merly called  bovine  colloid  but  which  we  now  call  conglutinin,  the 
intervention  of  which  explains  the  peculiarities  of  this  particular 
instance  of  hemolysis.  Without  going  into  details  as  to  this  latter 
work  (which  may  be  found  in  the  Centralblatt  fur  Bakteriologie, 
Vol.  49,  second  Heft),  I  may  content  myself  with  noting  that,  con- 
trary to  Ehrlich  and  Sachs'  opinion,  the  bovine  amboceptor  is  in  no 
way  abnormal  but  agrees  with  the  general  law,  which  is  that  the 
sensitizer  does  not  act  alone  in  absorbing  alexin,  but  only  the  com- 
plex, corpuscle-sensitizer.  Streng  has  since  continued  the  study  of 
bovine  serum  (see  Centralblatt  fur  Bakteriologie,  1909).  This 
author  has  made  some  very  interesting  observations  by  employing 
with  the  bovine  serum,  bacteria  instead  of  corpuscles,  which,  in  so 
far  as  the  theory  of  the  action  of  serum  is  concerned,  agree  perfectly 
with  the  experiments  on  corpuscles  which  we  had  performed.  My 
studies  with  Gay  on  the  antagonistic  power  of  serum,  and  Gengou's 
numerous  experiments  on  the  phenomenon  of  adsorption,  make 
more  evident  the  idea  that  fixation  of  alexin  is  in  reality  an  adsorp- 
tion phenomenon.  The  striking  analogies  so  far  as  the  action  of  the 
citrate  of  sodium  is  concerned,  between  the  fixation  of  alexin  or 
other  hemolysis  on  the  one  hand  and  the  adsorption  of  certain 
chemical  precipitates  on  the  other,  are  very  suggestive  and  it 
is  unnecessary  to  insist  on  them  further.  Which  of  the  argu- 
ments that  have  been  offered  in  favor  of  a  complementophi- 
lic  group  of  the  sensitizer  still  remain?  In  the  first  place  the 
existence  of  complementoids.  On  heating  certain  alexins  very 
carefully  (for  example  toward  52  degrees),  we  find  that  their  hemo- 
lytic  energy  is  much  decreased ;  but  alexins  that  have  been  altered 
in  this  manner  can  still  be  absorbed  by  sensitized  corpuscles.  As 
would  be  naturally  expected,  such  corpuscles  charged  with  com- 
plementoids, that  is  to  say  an  altered,  or  perhaps  better,  affected, 
alexin  are  thenceforth  incapable  of  fixing  active  alexin  for  the 
simple  reason  that  their  capacity  for  alexin  adsorption  is  not  with- 
out limits.  These  corpuscles,  having  been  saturated  with  weakened 
alexin,  subsequently  refuse  the  hemolytic  alexin  and  consequently 
remain  intact.  Gay  has  carefully  explained  this  phenomenon  in 
his  article  on  complementoids.  What  relation  indeed  have  these 


A  GENERAL  RESUME  OF  IMMUNITY.  513 

substances  with  the  theory  of  a  complementophilic  group?  If 
it  had  been  found  that  the  sensitizer  could  absorb  complements 
without  the  presence  of  antigen,  the  theory  would  have  been  forti- 
fied, but  no  such  thing  happens.  As  with  intact  alexin,  so  alexin 
that  has  been  altered  by  a  graduated  temperature  is  absorbed,  not 
by  the  sensitizer  alone,  but  only  by  the  complex,  corpuscle-sensi- 
tizer;  in  other  words,  the  general  law  holds. 

As  regards  the  arguments  furnished  by  a  study  of  cobra  venom 
and  lecithin,  there  is  nothing  to  prove  that  they  are  applicable  to 
sensitizers  and  alexin.  The  researches  of  Kyes  and  of  Neisser  and 
Friedemann  have  shown  clearly  that  the  venom,  although  in  itself 
unable  to  destroy  certain  corpuscles,  does  hemolyze  them  when 
added  to  lecithin;  in  other  words,  the  complex,  venom-lecithin  is 
remarkably  active.  But  what  reason  is  there  to  suppose  that  sen- 
sitizer and  alexin  necessarily  act  as  do  venom  and  lecithin?  There 
would  seem,  on  the  contrary,  no  parallel  between  them.  The  union 
of  venom  and  lecithin,  which  may  perfectly  well  be  a  simple  adsorp- 
tion phenomenon,  is  easily  demonstrable  experimentally,  whereas 
no  such  union  between  sensitizer  and  alexin  is  evident.  What  is 
more,  corpuscles  that  resist  the  action  of  venom  alone  but  are  he- 
molyzed  by  the  complex  of  lecithin  and  venom,  are  unable  to  fix 
venom  when  lecithin  is  not  present,  whereas  we  have  seen  that  the 
sensitizer  in  hemolytic  serum  can  always  be  absorbed  by  the  cor- 
puscles in  the  absence  of  alexin. 

Experiments  on  antisensitizers,  and  particularly  those  which  I 
published  in  1904,  have  been  made  use  of  by  Ehrlich  and  Sachs  as 
favoring  the  existence  of  a  complementophilic  group.  I  found  that 
sensitized  corpuscles,  when  mixed  with  a  suitable  antisensitizer,  lose 
their  power  of  absorbing  alexin;  in  other  words,  the  antisensitizer 
really  cures  the  corpuscles  since  it  annuls  the  effect  that  the  sensi- 
tizer has  produced.  From  this  observation  Ehrlich  and  Sachs  con- 
cluded that  the  antisensitizer  unites  with  a  complementophilic 
group  of  the  amboceptor  and  consequently  satisfies  its  affinities , 
According  to  these  authors  this  interpretation  is  the  only  one  which 
is  compatible  with  the  fact  that  the  antisensitizer  drives  out  the 
sensitizer  from  the  corpuscle.  It  is,  indeed,  true,  as  I  noted  in  my 
article,  that  the  antisensitizer  unites  with  the  corpuscle-sensitizer 
complex  but  does  not  eliminate  the  latter  substance.  It  simply 


514  STUDIES   IN  IMMUNITY. 

causes  the  complex  to  lose  its  affinity  for  alexin.  Adsorption  phe- 
nomena frequently  show  similar  precipitations  of  various  substances 
on  one  another;  for  example,  if  we  wash  a  rather  thick  suspension  of 
barium  sulphate  over  a  firm  paraffin  surface,  we  find  that  the 
paraffin  becomes  covered  with  a  delicate  white  layer  of  sulphate 
which  adheres  and  resists  washing.  Let  us  now  place  a  suspension 
of  red  blood  cells  on  such  a  paraffin  surface  that  has  been  covered 
with  barium  sulphate,  and  then  wash  with  salt  solution :  we  find  the 
corpuscles  adhere  to  the  sulphate  with  which  the  paraffin  is  covered ; 
the  surface  gradually  takes  a  reddish  color  which  resists  washing  in 
salt  solution.  The  paraffin  adsorbs  the  sulphate  which  in  its  turn 
adsorbs  the  corpuscles.*  It  is  evident  that  in  such  an  instance  the 
phenomenon  may  be  expressed  by  saying  that  the  sulphate  func- 
tions as  amboceptor  between  the  paraffin  and  the  corpuscle. 

But  are  we  always  obliged  to  conclude  that  when  a  property 
becomes  manifest  or  disappears  it  is  owing  to  the  integrity  of  or 
an  alteration  in  a  definite  atom  group?  When  a  sensitized  cor- 
puscle that  has  been  disintoxicated  by  antisensitizer  fails  to  fix 
alexin,  must  we  suppose  that  the  complementophilic  group  of  the 
sensitizer  is  thenceforth  saturated?  Such  an  interpretation,  in 
truth,  cannot  be  reconciled  with  fact;  experiment  shows  us  that  a 
given  antisensitizer,  for  example  the  one  furnished  by  guinea-pigs 
immunized  against  rabbit  serum,  neutralizes  all  the  sensitizers  from 
the  same  animal  species  (in  this  case  the  rabbit),  but  has  no  effect 
on  sensitizers  from  other  animal  species.  Studies  on  the  fixation  of 
alexin  show  that  the  sensitizers  of  different  animals  make  use  of  the 
same  alexin  in  bringing  about  either  hemolysis  or  bacteriolysis;  if 
we  were  to  use  Ehrlich's  terminology  we  should  be  forced  conse- 
quently to  conclude  that  they  have  identical  complementophilic 
groups.  According  to  Ehrlich's  interpretation  they  should  all  be 
neutralized  by  the  same  antisensitizer,  and  such  is  not  the  case. 
We  find,  moreover,  that  sensitized  corpuscles  treated  by  a  suitable 
antiserum  subsequently  resist,  whatever  alexin  be  employed.  And 
what  is  more,  the  phenomenon  of  alexin  fixation  appears  more  and 
more,  as  has  been  evidenced  in  the  preceding  pages,  to  be  an  adsorp- 

*  Gengou's  experiments,  as  is  well  known,  have  shown  that  barium  sulphate 
possesses  the  property  of  precipitating  itself  on  red  blood  cells  and  forming  clumps 
with  them. 


A  GENERAL  RESUME  OF  IMMUNITY.  515 

tion  phenomenon  which  does  not  depend  on  a  strictly  defined  atom 
group.  It  is  probable  that  the  properties  of  molecular  adhesion  in 
a  corpuscle-sensitizer  complex  may  be  modified  by  the  addition  of 
a  third  element,  the  antisensitizer,  and  that  the  tendency  for  alexin 
adsorption  will  disappear  correlatively.  In  the  same  way  the  tend- 
ency of  barium  sulphate  to  fix  itself  on  corpuscles  disappears  when 
the  particles  of  this  substance  have  been  previously  adsorbed  by 
gum,  by  certain  albuminoids,  or  by  sodium  citrate. 

And  finally  the  recent  researches  of  Dr.  Oswald  Streng  have 
shown  that  the  anti-alexins  act  in  the  same  way  as  the  antisensi- 
tizers.  Horse  alexin  becomes  fixed  easily  on  guinea-pigs'  corpuscles, 
but  inasmuch  as  it  is  only  slightly  hemolytic  it  does  not  destroy 
them.  These  corpuscles,  which  have  fixed  alexin,  are  henceforth 
capable  of  giving  a  reaction  with  the  conglutinin  of  bovine  serum; 
when  mixed  with  this  serum  (previously  heated  to  56  degrees)  they 
are  energetically  agglutinated.  If  we  take,  as  Sachs  did,  guinea-pig 
corpuscles  laden  with  horse  alexin,  and  treat  them  with  anti-alexin, 
that  is  to  say,  heated  serum  from  a  rabbit  immunized  against  horse 
serum,  and  then  test  them  with  bovine  serum,  we  obtain  no  con- 
glutinin reaction.  The  anti-alexin,  then,  has  neutralized  the  alexin 
which  has  been  fixed  to  the  corpuscles  and  has  cured  them  by  causing 
the  property  which  the  alexin  produced,  namely,  that  of  adsorbing 
conglutinin,  to  disappear.  And  yet  the  alexin  that  has  been  fixed 
with  corpuscles  has  no  free  group,  and  so  far  as  it  is  concerned  no 
explanation  similar  to  the  one  proposed  for  sensitizer  and  anti- 
sensitizer  by  Ehrlich  and  Sachs  can  be  accepted. 

*** 

Inasmuch  as  we  have  no  explanation  of  specificity  we  must  put 
aside  for  the  moment  the  question  as  to  why  such  and  such  an  anti- 
body unites  with  such  and  such  an  antigen.  But  even  if  the  under- 
lying reason  for  this  combination  escapes  us,  we  can  at  least  endeavor 
to  determine  how  this  combination  is  brought  about,  that  is  to  say, 
can  trace  its  development.  Attempts,  therefore,  have  been  made 
to  define  the  characters  of  the  reaction  and  to  determine  in  what 
category  of  phenomena  it  belongs.  The  reader  doubtless  knows  the 
opinions  which  have  been  offered  on  this  subject.  Certain  authors 
thought  that  this  reaction  should  be  likened  to  chemical  phenomena, 
properly  speaking,  as  they  exist  either  between  substances  endowed 


516  STUDIES  IN  IMMUNITY. 

with  an  energetic  affinity  for  one  another,  in  which  case  the  reaction 
is  complete  and  gives  rise  to  a  relatively  stable  compound  (Ehrlich), 
or  between  substances  with  weak  affinities,  in  which  case  the  com- 
bination is  only  partial  and  distinctly  reversible;  a  state  of  equili- 
brium in  other  words  would  be  established  between  the  fraction  of 
the  antigen  which  remains  free  and  the  one  which  enters  into  com- 
bination (Arrhenius  and  Madsen).  According  to  other  authors, 
and  I  believe  I  was  the  first  to  offer  this  opinion,  the  union  of  the 
antibody  with  the  antigen  depends  on  what  is  called  molecular 
adhesion  or  contact  affinity,  in  other  words  should  be  classed  in  the 
category  of  adsorption  phenomena.* 

In  this  connection  it  is  well  to  define  the  limit  of  the  debate  and 
avoid  misunderstandings.  The  phenomena  of  adsorption  are  fre- 
quently contrasted  with  true  chemical  phenomena,  but  it  is  not  the 
function  of  the  biologist  to  define  the  frontiers  of  physics  and  chem- 
istry. What  is  more,  this  task  is  no  easy  one,  since  the  most  au- 
thoritative physical  chemists,  as  Van  Bemmelen,  Nernst  and  many 
others,  themselves  consider  that  there  are  all  transitions  between 
data  of  adsorption  and  those  of  true  chemical  combination.  It  is, 
then,  superfluous  for  me  to  defend  myself  again,  as  I  have  already 
done  in  one  of  my  articles,  against  criticism  that  has  been  raised 
against  me  on  the  ground  that  I  deny  definitely  any  chemical 
character  to  the  union  of  antibody  and  antigen  by  classing  them 
among  adsorption  phenomena.  When  in  discussing  the  action  of 
sera  one  mentions,  indeed,  as  an  example,  the  dyeing  of  a  piece  of 
filter  paper  by  an  anilin  dye,  it  is  not  the  purpose  to  find  out  whether 

*  Inasmuch  as  it  is  not  possible  to  give  a  complete  historical  account  at  this 
time,  I  may  confine  myself  to  citing  what  Ulrich  Friedemann  has  published  in  the 
beginning  of  his  remarkable  work,  "Ueber  die  Fallung  von  Eiweiss  durch  andere 
Kolloi'de  und  Ihre  Beziehungen  zu  den  Immunkorperreaktionen,"  an  historical 
account  which  deals  with  the  first  articles  bearing  on  the  relation  of  adsorption 
to  immunity. 

"  Das  Studium  der  KolloTde  hat  bereits  vielfache  Aufschlusse  iiber  die  physi- 
kalisch-chemischen  Vorgange  bei  der  Immunitiitsreaktionen  gegeben.  Die 
Verbindungen  der  Immunkorper  wurden  mit  den  Adsorptionverbindungen  der 
Kolloide  vergleichen  (Bordet,  Landsteiner  und  Jagic,  Biltz,  Zaugger,  Much  und 
Liebert,  Pauli),  wahrend  sich  eine  bemerkenswerte  Ahnlichkeit  zwischen 
den  Fallungsreaktionen  der  Immunkorper  (Agglutination  und  Pr-izipitation) 
und  den  Gelbildungen  und  Prazipitations-erscheinungen  in  Kolloklaler  Losungen 
und  feiner  Suspensionen  herausstellte.  (Bordet,  Bechhold,  Neisser  und  ^riede- 
mann,  Biltz,  Landsteiner  und  Jagic,  Henri  und  Mitarbeiter,  Gengou.)" 


A  GENERAL  RESUME   QF   IMMUNITY.  517 

this  dyeing  is  a  physical  or  chemical  phenomenon,  but  simply  to 
determine  whether  there  are  analogies  in  the  mode  of  reaction 
between  the  paper  and  the  dye  on  the  one  hand,  and  antibody  and 
antigen  on  the  other. 

Our  knowledge  of  adsorption  owes  much  to  the  study  of  col- 
loids. Certain  authors,  indeed,  who  agree  with  me  in  recognizing 
the  great  importance  of  molecular  adhesion  in  serum  reactions, 
have  become  accustomed  to  say  that  the  union  of  the  antibody 
with  the  antigen  represents  a  reaction  between  two  colloids.  It 
seems  to  me  a  little  rash  to  define  so  clearly  and  I  should  prefer  to 
speak  of  this  union  as  an  adsorption  phenomenon.  Adsorption, 
indeed,  is  evident,  not  only  between  two  colloids  but  also  between  a 
colloid  or  suspended  particles  on  the  one  hand  and  a  substance  in 
true  solution  on  the  other,  and  although  it  is  very  probable  that 
antibodies  are  really  colloids  we  cannot  with  certainty  say  as  much 
of  the  antigens.  It  may  be  remarked  in  respect  to  colloids  that  too 
much  importance  has  frequently  been  attached  to  the  appearance 
of  agglutination  as  an  indication  of  the  formation  of  a  complex. 
The  agglutination  is  a  secondary  phenomenon  as  Neisser  and  Fried  e- 
mann,  Bechhold,  Biltz,  Henri  and  Girard  Mangin,  and  others  have 
correctly  stated,  and  its  manifestation  depends  in  large  measure  on 
certain  conditions  such  as  the  relative  proportions  of  the  two  sub- 
stances concerned,  temperature,  presence  of  electrolytes,  etc.  The 
principal  and  essential  fact  is  the  affinity  of  adhesion  produced  by 
the  union,  but  such  a  union  may  give  rise  quite  as  well  to  dissemina- 
tion, that  is,  a  more  stable  and  perfect  condition  of  emulsion,  as  to 
the  formation  of  flecks ;  in  his  researches  on  adsorption  by  chemical 
precipitates,  Gengou  has  insisted  on  this  fact  and  shown  that  slight 
physical  modifications  of  the  substances  concerned  without  any 
chemical  change  may  give  rise  to  dissemination  instead  of  clumping, 
or  the  reverse.  Forges  has  noted  similar  facts. 

Can  certain  characters,  such  as  reversibility  or  the  rapidity  of 
reaction,  be  called  upon  to  prove  whether  the  union  of  two  sub- 
stances belongs  or  not  in  the  category  of  adsorption  phenomena? 
In  this  respect  we  should  note,  particularly,  that  the  affinity  of 
adsorption  varies  greatly  in  different  cases  and  these  differences  of 
intensity  are  evident  by  the  more  or  less  rapid  union  and  the  more 
or  less  stable  resulting  complex.  As  Van  Bemmelen  has  mentioned, 


518  STUDIES  IN  IMMUNITY. 

the  power  of  adsorption  varies  with  each  adsorbing  substance  and 
each  adsorbed  substance.  It  depends,  certainly,  on  physical  quali- 
ties, but  also  on  the  chemical  nature  of  the  substances  concerned, 
and  would  appear  to  us  more  variable  still  if  we  could  measure  by 
delicate  methods  in  each  instance.  Certain  colors  act  on  substances 
like  paper  more  rapidly  than  others,  and  the  subsequent  decoloration 
of  the  paper  in  a  large  volume  of  water  is  sometimes  easy  and  some- 
times difficult.  In  the  adsorption  of  albuminous  substances  by 
inorganic  colloids  all  degrees  of  reversibility  are  found.  In  serum 
reactions  a  similar  irregularity  occurs.  Certain  antibodies  act  very 
rapidly  and  others  require  more  time  to  unite  with  their  antigens 
and  the  complexes  obtained  are  dissolved  with  very  unequal  facility. 
As  is  evident,  particularly  from  the  researches  of  Morgenroth,  that 
the  neutralizing  of  diphtheria  toxin  by  antitoxin  is  much  slower  than 
was  at  first  believed.  Certain  complexes,  such  as  those  formed  by 
agglutinins  or  sensitizers  with  bacteria  or  corpuscles,  or  by  certain 
toxins  with  their  antitoxins,  are,  if  not  completely,  at  least  dis- 
tinctly, reversible.  In  this  relation  I  may  recall  the  work  of  Hahn 
and  Tromsdorff,  Landsteiner,  Morgenroth,  Muir,  Madsen,  Otto  and 
Sachs.  But  it  should  be  noted  that,  in  general,  the  complexes  of 
antibody-antigen,  especially  when  they  have  been  standing  for  some 
time,  manifest  a  very  imperfect  reversibility,  distinctly  less  than 
Arrhenius  and  Madsen 's  thesis  would  demand.  In  the  same  way, 
in  general,  adsorption  phenomena  give  rise  to  incompletely  reversible 
complexes.  As  we  know  the  study  of  the  neutralization  of  toxins 
by  antitoxins  has  brought  out  the  fact,  particularly  owing  to  the 
important  researches  of  Ehrlich,  that  it  is  frequently  impossible  to 
prepare  mixtures  which  are  exactly  neutral.  It  is  likewise  known, 
so  far  as  diphtheria  toxins  and  antitoxins  are  concerned,  that  Ehrlich 
explains  this  fact  by  supposing  that  the  toxin  contains  in  reality 
several  poisons.  The  same  phenomenon,  however,  occurs  in  in- 
stances where  there  is  no  reason  for  supposing  that  such  a  com- 
plexity in  the  toxic  fluid  exists.  In  such  instances  there  is  a  priori 
no  reason  for  agreeing  with  the  thesis  of  Arrhenius  and  Madsen  in 
accordance  with  which  the  reaction  is  incomplete  and  leaves  a  cer- 
tain amount  of  the  toxin  free,  or  with  my  own  theory  in  accordance 
with  which  the  molecules  of  toxin  and  antitoxin  may  unite  in  vari- 
able proportions  according  to  the  respective  quantities  of  the  two 


A  GENERAL  RESUME  .OF   IMMUNITY.  519 

substances  in  the  mixture.  A  comparison  with  a  process  of  dyeing 
is  very  apt;  the  molecules  of  the  toxin  would  " stain"  more  or  less 
deeply  by  the  antitoxin  molecules,  and  the  complexes  that  result 
in  the  various  instances  are  less  toxic  in  proportion  as  they  contain 
more  antitoxin  and  less  toxin.  I  brought  out  this  point  of  view 
clearly  in  1903  and  discussed  all  the  details  of  it  so  that  no  further 
insistence  on  the  subject  is  necessary.  It  may  be  added  that  this 
interpretation  has  met  with  the  support  of  various  writers,  among 
whom  may  be  mentioned  Grassberger  and  Schattenfroh,  Biltz  and 
Pauli,  who  have  accepted  it  in  its  entirety. 

The  essential  point  of  difference  between  these  different  concep- 
tions evidently  lies  in  attributing  a  different  composition  to  the  mix- 
ture of  toxin  and  antitoxin  and  particularly  to  such  mixtures  as 
contain  too  small  a  dose  of  the  antidote  to  bring  about  complete 
neutralization.  In  order  to  simplify  it  we  may  choose  a  toxic  fluid 
which  contains  a  single  poison  and  add  to  it  a  relatively  small 
amount  of  suitable  antitoxin.  In  such  a  case  are  we  to  suppose 
that  the  mixture  includes  both  well  neutralized  toxin  and  an  excess 
of  a  free  and  intact  toxin,  or  are  we  rather  to  believe,  as  I  thought, 
that  the  antitoxin  is  shared  by  the  totality  of  the  toxin  present, 
but  saturates  it  only  partially  in  the  same  way  that  a  small 
amount  of  dye  does  a  large  quantity  of  the  substance  to  be  dyed,  by 
spreading  over  the  whole  of  it  and  staining  it  faintly?  The  complex 
thus  obtained,  which  is  weak  in  antitoxin,  would  still  have  certain 
toxic  properties  and  would  act  like  toxon. 

But  how,  it  may  be  objected,  can  we  determine  with  certainty 
whether  the  toxic  activity  of  such  a  mixture  is  due  to  a  certain 
amount  of  intact  toxin  or  the  presence  of  a  partially  saturated  com- 
plex? It  is  here  that  we  must  distinguish  very  carefully  in  the 
effects  of  a  poison  between  quality  and  quantity.  I  may  recall 
the  observations  which  I  offered  in  1903  that  showed  that  a  hemoly- 
sin  may  manifest  toxicity  in  two  different  ways;  when  employed 
in  a  small  dose  without  being  in  any  way  affected  by  an  antitoxin, 
it  hemolyzes  a  small  amount  of  corpuscles  rapidly,  but  owing  to 
the  fact  that  it  is  not  present  in  sufficient  quantities  it  cannot, 
destroy  many  of  them.  On  the  other  hand,  a  large  dose  of  the  same 
hemolysin  to  which  a  little  of  its  antitoxin  has  been  added  and 
which  has  been  transformed  thereby,  according  to  my  idea,  into  an 


520  STUDIES  IN  IMMUNITY. 

incompletely  saturated  complex,  can  hemolyze  a  large  amount  of 
corpuscles  but  produces  hemolysis  very  slowly,  even  if  a  very  small 
amount  of  the  corpuscles  is  added.  It  would  seem,  then,  as  if  such 
a  mixture  included  a  strong  dose  of  poison,  the  activity  of  which  is 
distinctly  depressed  owing  to  the  addition  of  a  little  antitoxin,  and 
the  result  of  the  experiment  is  in  no  way  compatible  with  the  idea 
that  such  a  mixture  contains,  in  addition  to  perfectly  neutralized 
hemolysin,  a  certain  excess  of  perfectly  intact  hemolysin.  But 
there  is  another  way  of  proving  that  an  antigen  can  unite  with  the 
antibody  in  variable  proportions  and  so  form  complexes  of  varying 
constitution  in  accordance  with  the  amounts  of  each  substance 
present.  It  may  be  shown  that  such  complexes  do  not  react  in 
the  same  way  to  certain  agents  such  as  heat.  It  is  perfectly  evident 
from  the  observations  of  Eisenberg  and  Volk,  that  the  union  of  the 
agglutinin  with  the  agglutinable  substance  of  bacteria  takes  place 
in  variable  proportions.  Landsteiner  and  Jagic  have  confirmed  this 
idea  by  showing  that  the  complexes  obtained,  which  differ  in  the 
relative  proportions  of  each  constituent,  show  different  resistance 
to  heat.*  The  brilliant  researches  of  Grassberger  and  Schattenfroh 
on  the  toxin  of  symptomatic  anthrax,  are  very  instructive  in  this 
connection.  When  we  mix  a  certain  dose  of  the  toxin,  either  with 
little  or  with  much  antitoxin,  we  obtain  complexes  of  toxin-anti- 
toxin, which  vary,  as  is  shown  by  their  varying  reaction  to  heat. 
It  is  to  be  noted  in  the  first  place  that  the  toxin  in  question  is  ther- 
molabile,  whereas  the  antitoxin  resists  heat  much  better.  The 
complexes  containing  a  large  amount  of  antitoxin  enjoy  the  prop- 
erties of  this  substance,  that  is  to  say,  they  resist  heat  much  better 
than  do  complexes  which  are  weak  in  antitoxin;  these  latter  mix- 
tures are  decomposed  at  a  given  temperature,  and  the  poison  is 
destroyed  so  that  the  antitoxin  may  be  recovered.  These  authors 
have  further  shown  themselves  partisans  of  the  idea  that  antitoxin 
unites  with  toxin  in  variable  proportions.  They  have  been  able  to 
prove  that  their  poison  absorbs  much  more  antitoxin  than  is 
necessary  to  destroy  its  entire  toxicity  and  forms  a  stable  complex 

*  I  noted  in  1896  (Mode  of  action  of  preventive  sera),  that  heating  toward 
50  degrees  brings  about  a  real  disagglutination  of  the  substances  which  have  been 
agglutinated.  This  is  due  to  the  fact,  as  Landsteiner  and  his  collaborators  have 
shown,  that  the  complex  is  dissociated.  What  is  more,  the  ease  of  this  dissociation 
varies  with  the  amount  of  agglutinin  present. 


A   GENERAL  RESUME   OF   IMMUNITY.  521 

with  it.  They  have  also  found  that  dilution  renders  neutralization 
much  more  difficult  and  that  different  mixtures  are  obtained  depend- 
ing on  whether  they  mix  the  toxin  and  the  antitoxin,  after  diluting 
them,  or  dilute  the  toxin-antitoxin  mixture.  This  fact  is  evi- 
dently not  in  favor  of  the  Arrhenius-Madsen  theory,  according  to 
which  the  same  state  of  equilibrium  should  exist  in  both  instances 
owing  to  reversibility,  and  the  same  fraction  of  the  toxin  of  necessity 
remain  free. 

The  fact  which  lends  significance  to  the  researches  of  Grassberger 
and  Schattenfroh,  and  which  prevents  their  applying  Ehrlich's 
interpretation,  is  that  the  toxic  fluid  which  they  employ  contains 
in  reality  a  single  poison;  there  is  no  reason  for  assuming  the  exis- 
tence of  toxoids,  inasmuch  as  the  toxic  power  of  the  poison  shows 
itself  constantly  parallel  to  its  neutralizing  power  for  antitoxin. 

Among  the  facts  which  have  most  contributed  to  strengthen  the 
idea  of  a  union  in  variable  proportions  (an  idea  which  is  so  highly 
compatible  with  the  adsorption  theory)  and  which,  moreover,  have 
shown  that  the  complexes  obtained  frequently  manifest  only  a  very 
incomplete  reversibility,  there  should  be  mentioned  in  particular 
those  which  have  been  observed  by  employing  the  method  which  I 
used  for  hemolysins  in  1900  and  which  may  be  called  "the  method 
of  addition  of  the  antigen  to  the  antibody  in  single  or  in  divided 
doses."  A  given  volume  of  hemolytic  serum  may  destroy  more 
or  less  corpuscles  in  accordance  with  their  introduction  in  one  or 
several  doses  separated  by  sufficient  intervals.  As  we  know,  other 
authors  have  since  performed  similar  experiments  with  the  same 
results,  employing  antitoxin  and  toxin  in  place  of  hemolysin  and 
corpuscles  (Danysz,  von  Dungern,  and  Sachs)  or  using  bacteria  and 
agglutinins  (Craw).  It  seems  to  me  proper  in  this  connection  to 
mention  the  comparison  with  dyeing  phenomena,  and  on  this 
point  I  agree  entirely  with  Craw.  If  a  large  piece  of  filter  paper 
is  placed  in  a  certain  volume  of  sufficiently  diluted  dye  it  takes  a 
uniform  shade  of  intensity;  if,  on  the  other  hand,  the  same  sized 
piece  of  paper  is  cut  up  in  pieces  and  added  in  fragments,  the  first 
pieces  are  stained  deeply  and  the  last  find  no  color  left.  In  the 
same  way,  on  adding  toxin  to  antitoxin  in  divided  doses  the  last 
portions  of  the  poison  cannot  be  neutralized  as  the  first  are  super- 
saturated with  antitoxin.  When  the  entire  mixture  is  made  at 


522  STUDIES   IX   IMMUNITY. 

once,  on  the  contrary,  the  antitoxin  is  spread  over  all  the  toxic 
molecules  and  a  complex  is  obtained  which  contains  an  even  pro- 
portion of  the  antidote,  and  which,  consequently,  is  not  as  fatal  as 
even  a  small  dose  of  free  toxin. 

It  is  an  extraordinary  thing  that,  so  far  as  precipitins  and  its  pre- 
cipitable  antigens  are  concerned,  this  interpretation  is  not  contested, 
even  by  such  writers  as  von  Dungern,  who  are,  in  general,  favorable 
to  Ehrlich's  ideas.  As  Halban  and  Landsteiner,  Eisenberg,  von 
Dungern,  and  Miiller,  in  particular,  have  seen,  if  we  mix  a  precip- 
itable  serum  "A"  with  its  appropriate  precipitating  serum  "B,"  an 
excess  of  the  precipitable  substance  inhibits  the  occurrence  of  a 
precipitate.  For  example,  it  is  found  that  in  a  mixture  of  one 
volume  of  serum  "A"  with  one  volume  of  serum  "B,"  a  cloudiness 
appears,  but  a  mixture  composed  of  two  volumes  of  "A"  and  one 
volume  of  "B,"  remains  translucent.  The  almost  obvious  explana- 
tion is  that  the  molecules  of  "A"  are  not  susceptible  to  precipita- 
tion unless  they  have  fixed  a  sufficient  number  of  molecules  of 
"B."  Such,  however,  is  the  condition  in  the  first  mixture.  In  the 
second  mixture  the  molecules  of  "B"  are  shared  among  all  the 
molecules  of  "A"  which  are  in  too  great  a  number  to  permit  their 
perfect  separation  and  consequently  they  are  not  precipitated. 
In  accordance,  then,  with  the  proportions  of  constituent  parts 
different  complexes  may  be  obtained,  and  this  fact  comes  out 
quite  clearly  in  the  experiment  of  the  addition  in  a  single  or  in 
divided  doses.  If  we  add  one  volume  of  "B"  to  two  volumes  of 
"A,"  the  mixture  remains  limpid,  even  after  a  considerable  time. 
If,  on  the  other  hand,  we  add  one  volume  of  "B"  to  one  volume  of 
"A,"  a  precipitate  immediately  appears.  When  the  precipitate  is 
completely  formed  we  may  add  another  volume  of  "A"  so  as  to 
make  the  mixture  correspond  to  the  preceding  one.  The  precipi- 
tate does  not  disappear,  but  remains  indefinitely  intact  and  so  we 
have  obtained  two  mixtures  composed  of  the  same  sera  in  the  same 
proportions  but  presenting  entirely  different  aspects. 

Does  not  this  experiment  offer  most  satisfactory  analogies  with 
the  one  on  coloring  filter  paper  with  a  dye  to  which  I  have  just 
referred?  The  objection  may  be  raised  that,  although  true  as  far 
as  precipitins  are  concerned,  it  may  not  be  applicable  to  antitoxins. 
Such  an  objection,  however,  would  be  to  deny  any  character  of 


A  GENERAL  RESUME   OF   IMMUNITY.  523 

unity  in  the  laws  which  govern  the  action  of  antibodies;  it  would 
be  to  distribute  them  into  divisions,  separated  by  impassable  bar- 
riers, and  I  have  already  mentioned  in  speaking  of  Ehrlich's  classifi- 
cation how  inacceptable  such  a  system  is.  Certain  antigens  are  toxic 
and  certain  others  have  a  greater  tendency  toward  precipitation 
than  others;  such  differences  between  the  antigens,  however,  do 
not  prevent  us  from  supposing  that  their  antibodies  are  very  similar 
and  that  their  action  is  subject  to  the  same  interpretation  in  the 
various  instances. 

A  phenomenon  which  has  justly  attracted  the  attention  of  experi- 
menters is  the  increasing  stability  of  complexes  of  antibody-antigen 
following  their  formation.  It  would  seem  as  if  these  complexes, 
which  are  easily  dissociable  in  the  beginning,  subsequently  become 
consolidated  in  some  manner  so  as  to  resist  decomposing  influences 
better.  Facts  of  this  sort  have  been  noted  by  Landsteiner  and 
Jagic  with  agglutinins,  and  by  von  Dungern,  Sachs,  and  Otto 
and  Sachs  with  the  antitoxins  of  certain  poisons  such  as  the  toxin 
of  botulismus,  arachnolysin  and  the  like.  Facts  of  this  nature,  it 
seems  to  me,  should  be  interpreted  in  accordance  with  the  adsorp- 
tion theory.  As  Nernst  has  noted,  certain  dyes  as  they  unite 
more  and  more  intimately  with  the  object  which  they  stain,  lose 
correspondingly  their  adhesion  for  the  surrounding  fluid;  when 
they  have  become  fixed  they  show  a  distinct  tendency  to  become 
insoluble.  It  seems  that  we  may  treat  as  an  analogy  the  fact 
that  various  albuminous  fluids  treated  by  such  agents  as  alcohol 
give  precipitates  which  redissolve  easily  in  water  when  they  have 
been  recently  formed,  but  become  more  and  more  insoluble  when 
kept.  The  adhesion  which  unites  the  albuminous  molecules  with 
one  another  and  gives  them  a  solid  state  becomes  preponderating 
and  opposes  the  dissemination  which  the  contrary  effect,  the 
adhesion  for  water,  would  tend  to  produce.  The  consolidation  of 
complexes  of  antibody  and  antigen  is  apparently  a  phenomenon 
of  the  same  class. 

The  quantities  of  antibodies  which  antigens  can  fix  depending  on 
their  concentration,  have  been  determined  particularly  with  aggluti- 
nation phenomena  by  the  important  researches  of  Eisenberg  and 
Volk.  The  greater  the  concentration  of  the  antibody  the  more  the 
fixation  of  the  antigen,  but  the  quantity  fixed  does  not  increase  so 


524  STUDIES   IN  IMMUNITY. 

rapidly  as  the  concentration;  the  antigen  loses  its  avidity  for  anti- 
body in  proportion  to  the  amount  of  it  that  has  been  fixed.  This, 
according  to  general  opinions,  is  the  way  in  which  adsorption 
phenomena  act. 

It  is  evident  from  an  examination  of  these  facts  that  the  presiding 
force  in  the  union  of  antigens  with  their  antibodies  is  the  same  as 
the  one  which  produces  the  reactions  that  have  been  grouped  under 
the  name  of  adsorption  phenomena.  But  is  this  force  or  affinity 
of  adsorption  sufficiently  elective  to  permit  one  to  attribute  to  it 
alone  serum  reactions,  the  most  striking  characteristic  of  which  is 
their  remarkable  specificity?  It  has  become  more  and  more  certain 
that  adsorbing  substances  manifest  toward  adsorbable  substances 
which  are  mixed  with  them,  tendencies  of  attraction  of  very  unequal 
intensity  and  consequently  the  affinity  of  adsorption  is  similar  to 
a  true  chemical  affinity;  both,  in  other  words,  are  elective.  In 
both  instances  a  struggle  may  take  place  between  two  substances 
for  the  possession  of  a  third.  In  the  same  way  fixation  of  one  sub- 
stance on  another  may  be  inhibited  by  a  monopoly  of  the  first  by  a 
third  substance  which  thus  protects  the  second.  For  example,  the 
albuminous  substances  of  blood  protect  red  blood  cells  from  soap 
(Meyer),  or  even  against  the  hemolysin  of  eel  serum  (Frouin); 
sodium  citrate  protects  corpuscles  from  the  agglutinating  and 
hemolytic  effect  of  barium  sulphate  (Gengou),  and  so  on.  The 
lecithin  which  is  present  in  bovine  serum  is  probably  united  with 
another  substance,  without  doubt  of  an  albuminous  nature,  for  if  it 
were  not  so  agglutination  would  take  place  in  this  serum  itself.* 
In  all  instances  a  real  struggle  occurs  between  adsorption  affinities; 
similar  facts  have  also  been  found  with  animal  charcoal.  Substi- 
tutions may  also  be  noted.  Barium  sulphate  manifests  avidity  for 
mucin  and  for  sodium  citrate.  When  mixed  with  lecithin  it  clumps 
in  large  masses,  and  on  adding  sodium  citrate  to  them  they  rapidly 
dissolve  and  the  mucin  is  liberated  (Gengou),  in  other  words,  the 
sulphate  prefers  citrate  to  the  mucin. 

Certain  substances  in  the  category  of  lipoids  which  may  be  ob- 

*  As  we  know,  Toyosumi  found  that  bovine  serum  has  the  power  of  agglutinat- 
ing an  aqueous  emulsion  of  lecithin.  Sleeswijk,and  I  have  found  that  this  is  also 
true  even  with  an  extract  (by  means  of  methyl  alcohol)  of  the  lecithin  from  dried 
bovine  serum. 


A  GENERAL  RESUME  OF  IMMUNITY.  525 

tained  as  Landsteiner,  Pick  and  other  scientists  have  done  by 
extracting  corpuscles  or  bacteria  in  colloidal  suspension,  are  inter- 
esting from  the  standpoint  of  the  selective  action  of  serum.  Slees- 
wijk  and  I  have  recently  studied  the  action  of  hen  serum  on  the 
lipoids  extracted  from  red  blood  cells  by  methyl  alcohol  and 
subsequently  made  into  the  form  of  an  emulsion  in  salt  solution. 
We  found,  for  example,  that  hen  serum  agglutinates  the  lipoids 
extracted  from  rabbit  corpuscles  energetically,  but  has  very  little 
effect  on  those  from  bovine  corpuscles.  The  agglutination  of 
an  emulsion  of  lipoids  is  certainly  an  adsorption  phenomenon.  In 
this  case  adsorption  affinity,  of  varying  intensity  in  accordance 
with  the  substances  employed,  may  certainly  be  stated  as  the  cause 
of  the  differences  observed  when  a  given  serum  is  mixed  with  differ- 
ent bacteria  or  corpuscles.  And,  moreover,  the  adhesion  properties 
are  a  function  not  only  depending  on  the  chemical  constitution  but 
also  on  the  physical  state  of  the  substances.  It  is  easily  conceivable, 
then,  that  the  aptitude  of  the  antigen  to  react  with  the  antibody  may 
disappear  when  affected  by  slight  alterations  or  simple  physical 
modifications.  It  is  easy  to  understand,  then,  why,  in  Obermeyer 
and  Pick's  experiments,  the  serum  obtained  by  immunizing  animals 
against  certain  albuminous  substances  differed  from  the  serum 
obtained  by  immunizing  animals  against  the  same  substances  pre- 
viously heated.  In  brief,  although  an  explanation  of  the  specificity 
of  sera  is  not  yetxlearly  evident,  we  have  at  least  the  right  to  con- 
ceive of  it  as  harmonious  with  the  conception  of  adsorption  affinity 
as  the  essential  factor. 

*** 

This  important  problem  of  specificity  must  appeal  to  all  bacteri- 
ologists and  I  have  recently  begun  to  study  it  experimentally 
with  my  collaborator,  Sleeswijk.  Inasmuch  as  our  researches  are 
not  quite  finished,  I  may  content  myself  with  mentioning  certain 
facts.  The  problem  of  specificity  may,  obviously,  be  regarded  from 
two  different  aspects.  When,  for  example,  we  study,  an  antibacterial 
serum  we  wonder,  on  the  one  hand,  why  the  serum  is  specific,  and  on 
the  other  hand,  why  the  organism  allows  itself  to  be  specifically 
affected.  In  other  words,  both  the  antibody  and  the  affected  ele- 
ment must  be  considered.  I  cannot  deal  here  with  the  second  half 
of  the  question. 


526  STUDIES  IN  IMMUNITY. 

We  know  how  to  recognize  a  given  bacterium  by  means  of  a 
specific  serum.  But  is  the  character  which  this  organism  possesses 
of  reacting  with  the  appropriate  serum  constantly  present?  May 
it  not  disappear  when  the  organism  has  been  subjected  to  certain 
exigencies?  What,  for  example,  is  the  effect  oi  change  in  culture 
medium?  As  early  as  1896  Metchnikoff  and  I  noted  that  the 
cholera  vibrio  as  a  result  of  certain  vicissitudes,  such  as  remaining 
with  leucocytes,  comes  to  resist  the  agglutinating  effect  of  cholera 
serum  markedly,  and  since  that  time  many  analogous  instances 
have  been  collected.  The  remarkable  work  of  Grassberger  and 
Schattenfroh  on  symptomatic  anthrax  gives  most  interesting  infor- 
mation on  this  point.  These  authors  have  found  that  a  serum  that 
is  capable  of  agglutinating  the  organism  when  it  is  cultivated  in  a 
given  medium,  has  scarcely  any  agglutinating  effect  on  it  when 
grown  in  a  different  medium. 

We  could  understand  this  fact  by  supposing  that  the  organism 
has,  on  account  of  certain  vital  reactions,  succeeded  in  resisting  the 
effects  of  the  agglutinin,  although  it  has  kept  its  property  of  com- 
bining with  it;  in  other  words,  we  would  be  dealing  with  the  simple 
phenomenon  of  adaptation  to  a  harmful  effect  by  the  formation  of 
a  refractory  condition.  But  as  we  shall  see,  the  modification  is 
still  more  profound.  As  a  matter  of  fact,  owing  to  certain  con- 
ditions of  life,  the  organism  may  completely  lose  the  antigen, 
which  combines  with  the  antibody  and  which  is  its  principal  char- 
acteristic. In  other  words,  the  distinctive  sign  in  serum  diagnosis 
may  be  lost.  As  far  as  the  action  of  the  serum  is  concerned,  the 
organism  thenceforth  acts  as  if  it  belonged  to  a  different  species, 
which  proves  distinctly  that  specific  sera  (or  at  least  the  agglutinins ) 
do  not  act  primarily  on  those  substances  of  the  bacteria  which  are 
essential  to  their  life  and  characteristic  of  their  particular  species, 
but  on  certain  of  the  accessory  substances  of  the  organism  which 
may  occur,  but  the  existence  of  which  is  in  no  way  part  of  the  fixed 
hereditary  character,  which  gives  the  living  organism  its  particular 
appearance  and  autonomy. 

The  whooping-cough  bacillus  is  very  suitable  for  a  study  of  this 
sort.  We  find  that  this  organism  is  very  variable,  not  only  as 
regards  the  appearance  of  its  growth,  but,  more  important  still,  as 
regards  its  antigen,  in  accordance  with  the  conditions  under  which 


A  GENERAL  RESUME  OF  IMMUNITY.  527 

it  has  been  grown.  It  develops  readily  on  the  medium  that  is  rich 
in  defibrinated  blood,  as  already  described  by  Gengou  and  myself 
in  our  first  article  on  whooping  cough.  It  may  be  taught  to  grow 
on  ordinary  agar,  in  which  instance  it  gives  a  thick  and  rather 
coherent  layer.  The  two  varieties  of  organisms  obtained  in  this 
manner,  although  coming  from  a  single  original  colony,  give  rise,  on 
immunizing  animals,  to  two  different  sera.  We  may  consider  the 
serum  of  a  rabbit  that  has  been  immunized  against  the  organism 
grown  on  ordinary  agar.  It  is  found  that  the  serum  agglutinates 
these  organisms  energetically,  but  has  no  clumping  effect  on  a  cul- 
ture of  whooping-cough  bacillus  on  the  other  medium  containing 
defibrinated  blood.  On  the  other  hand,  if  we  test  the  serum  of  a 
rabbit  that  has  been  immunized  against  the  organism  grown  on 
blood  media,  we  find  that  it  agglutinates  both  races  of  bacteria.  A 
careful  study  of  this  phenomena  brings  out  the  fact  that  two  definite 
agglutinins  affecting  different  antigens  are  present  in  different  pro- 
portions. One  of  these  antigens,  which  is  present  in  large  amount 
in  the  organism  that  has  been  developed  on  blood,  is  not  to  be  found 
in  the  organisms  grown  on  agar.  To  demonstrate  this  antigen  we 
take  a  small  amount  of  the  serum  and  mix  it  with  an  excess  of  bac- 
teria grown  on  agar.  A  few  hours  later  we  centrifugalize  and 
decant  the  supernatant  fluid;  we  find  that  this  fluid  no  longer 
affects  the  same  bacteria  but  has  retained  entirely  its  agglutinating 
effect  for  the  other  strain  cultivated  on  defibrinated  blood. 

Horses  immunized  against  the  organism  grown  on  blood  *  furnish 
a  serum  with  an  extremely  marked  agglutinating  property  for 
this  strain  of  organism,  but  practically  no  active  agglutinin  for  the 
organism  grown  on  agar.  The  serum,  when  treated  with  even  a 
considerable  amount  of  these  latter  organisms,  even  after  prolonged 
contact,  is  found  to  have  lost  none  of  its  specific  effect  for  cultures 
grown  on  defibrinated  blood.  The  effect  on  these  latter  cultures, 
then,  is  due  to  an  antibody  which  does  not  find  a  suitable  antigen 
in  bacteria  of  the  same  species  grown  in  a  different  culture  medium. 

The  two  strains  that  we  have  considered  may  be  distinguished 

from  other  standpoints  on  which  I  shall  not  insist  for  the  moment, 

particularly  as  regards  their  sensitivity  to  the  antibodies  of  normal 

sera.     I  may  add,  however,  that  the  reaction  of  alexin  fixation 

*  Horse  blood  is  used  for  these  cultures. 


528  STUDIES   IN   IMMUNITY. 

does  not  serve  to  differentiate  them  as  clearly  as  agglutination 
does.  This  fact  may  be  compared  with  the  observations  of 
various  authors  in  accordance  with  which  the  reaction  of  fixation 
does  not  show  as  strict  a  specificity  as  the  reaction  of  agglutination. 

In  so  far  as  the  production  of  antigens  which  are  sensitive  to 
agglutinating  antibodies  is  concerned,  we  may  conclude  that  the 
culture  medium  may  be  of  very  great  importance.  It  may  still 
further  be  shown  that  these  two  strains  of  organisms  may  be  trans- 
formed into  one  another  very  rapidly  by  changing  the  culture 
medium.  Thus,  following  two  successive  generations  on  blood 
medium,  the  organism  that  has  been  previously  cultivated  on  agar 
recovers  the  faculty  of  producing  the  antigen  which  is  character- 
istic of  blood  cultures  and  consequently  becomes  agglutinable  by 
the  serum  of  a  horse  that  has  been  immunized  against  such  cultures. 

I  cannot  go  further  into  these  researches,  the  details  of  which 
would  exceed  the  limits  of  this  short  summary,  but  the  little  that  I 
have  said  suffices  to  justify  experimentally  the  opinion  which  is  also 
expressed  in  the  work  of  Grassberger  and  Schattenfroh,  that  cul- 
tures of  bacteria  developed  on  culture  media  which  differ  too  much 
from  the  body  fluids  (for  example,  grown  on  bouillon  or  agar  steri- 
lized in  the  autoclave),  are  not  suitable,  perhaps,  to  be  employed  in 
immunizing  animals  for  the  production  of  therapeutic  sera.  It  is, 
perhaps,  possible  that  bacteria  grown  on  these  two  artificial  culture 
media  fail  to  form  all  the  antigens  which  they  produce  in  the  animal 
body  during  infection  and  which  would  be  affected  by  suitable 
antibodies. 

*** 

I  may  conclude  this  brief  resume*  at  this  point.  Although  I  have 
discussed  in  some  detail  the  properties  of  sera,  I  have  not  taken  up 
at  all  the  essential  topic  of  phagocytosis.  The  importance  of  this 
phenomenon  in  the  defence  of  the  animal  body,  which  was  so 
much  combated  fifteen  years  ago,  has  no  need,  at  the  present  day, 
of  emphasis.  We  have  long  since  passed  the  time  when  the  exact 
observations  and  the  decisive  experiments  of  my  former  master 
and  present  friend,  Elie  Metchnikoff,  met  with  warm  but  often 
superficial  opposition  from  those  scientists  who  were  too  exclu- 
sively preoccupied  by  the  antibacterial  properties  of  the  body  fluids. 
Many  facts  which  have  long  been  known,  but  the  significance  of 


A  GENERAL  RESUME  OF  IMMUNITY.  529 

which  has  not  been  appreciated,  have  been  confirmed  and  re-studied 
now  that  there  is  a  generalizing  acceptance  of  the  value  of  phagocytic 
defense.  And  this  is  particularly  the  case  with  the  numerous  facts 
which  we  owe  to  Metchnikoff.  Such  is  the  case  also  with  certain 
of  the  facts  that  have  been  mentioned  in  various  articles  of  this 
volume,  particularly  as  regards  negative  chemiotaxis,  the  phenom- 
enon of  adaptation  which  bacteria  employ  to  protect  themselves 
against  phagocytosis,  and  the  visible  index  of  which  consists  in  the 
appearance  of  a  capsule,  the  manner  in  which  leucocytes  act  with 
certain  poisons,  toxins,  or  alkaloids  like  quinine,  and  the  like.  The 
fundamental  importance  of  phagocytosis  is  to-day  universally  ad- 
mitted and  is  moreover  evidenced  by  the  large  number  of  articles 
which  deal  with  means  of  computing  the  intensity  of  this  phenom- 
enon in  normal,  infected,  or  immunized  animals. 


INDEX   OF  AUTHORITIES   QUOTED. 


Afanassiew,  478. 

Amberger.     See  Paal. 

Aron,  422. 

Arrhenius  and  Madsen,  295,  516,  518, 

521. 

Arthus,  425,  426,  427,  429. 
Aschoff,  245. 
Azalier.     See  de  Coninck. 

Bail,  238,  500. 

Baratt,  397. 

Bauer.     See  Sachs. 

Bayliss,  437. 

Bechhold,  422,  432,  500,  516,  517. 

v.  Behring,  77. 

van  Bemmelen,  415,  418,  422,  516,  517. 

Besredka,  489. 

Biltz,  421,  516,  517,  519. 

Binot,  223,  469. 

Bordet,  Charles.     See  Massart. 

Bordet,  208,  241,  242,  243,  246,  248, 

252,  257,  258,  333,  334,  336,  358, 

363,  365,  389,  398,  403,  438,  441, 

467,  516. 
Bordet  and  Gay,  393,  403,  434,  442, 

443,  445,  446,  447,  448,  449,  451, 

452,  453,  460,  461,  509,  511,  512. 
Bordet   and    Gengou,    245,    246,    248, 

346,  398,  462,  464,  465,  467,  468, 

493,  508,  527. 

Bordet  and  Sleeswijk,  524,  525. 
Bordet  and  Streng,  511. 
Bredig,  314. 
Browning.     See  Muir. 
de  Bruyn,  415,  420. 
Buchner,  25,   51,   136,   153,   188,  213, 

219,  235,  242,  277. 
Buxton,  366. 

Camus  and  Gley,  176,  197,  281. 
Cantacuzene,  19,  35. 


Canthack,  24. 

Charrin  and  Roger,  18,  88. 

Cherry.     See  Martin. 

Cohen,  479,  483. 

Cohen,  E.,  415. 

de  Coninck  and  Azalier,  424. 

Craw,  441,  521. 

Czaplewski  and  Henzel,  478. 

Danysz,  35,  438,  441,  521. 

Dean,  397. 

Dembinski,  464,  466,  468. 

Denys,  25. 

Denys  and  Leclef,  120,  123,  125,  133, 

390. 

Denys  and  Marc  hand,  118. 
Dieudonne",  256. 
D  incur,  147,  148,  150. 
Douglas.     See  Wright. 
Duclaux,  153,  154,  157,  158,  506. 
Dunbar,  60,  77. 

v.  Dungern,  211,  441,  521,  522,  523. 
Durham,  105.     See  also  Gruber. 
Dujardin-Beaumetz,  219. 
Dyer  and  Madsen,  278,  279. 

Ehrlich,  22,  33,  259,  264,  266,  267, 
279,  337,  340,  383,  403,  410,  458,  497, 
498,  501,  508,  514,  516,  518,  521,  522. 

Ehrlich  and  Morgenroth,  161,  190,  191, 
210,  215,  229,  230,  231,  232,  234, 
235,  237,  240,  242,  243,  253,  288, 
294,  298.  302,  303,  333,  334,  336, 
364,  365,  366,  368,  371,  510,  511. 

Ehrlich  and  Sachs,  340,  366,  368,  369, 
370,  372,  374,  377,  378,  379,  380, 
381,  382,  383,  384,  385,  387,  388, 
393,  442,  443,  444,  445,  446,  449, 
452,  453,  456,  457,  461,  511,  513,  515. 

Eisenbcrg.     See  Kraus. 


531 


532 


INDEX  OF   AUTHORITIES. 


Eisenberg  and  Volk,  262,  263,  418,  500, 

520,  522,  523. 
Elmassian,  477. 

Englemann  and  Gaidukow,  498. 
En-era,  1. 

Fally,  492. 

Fassin,  479. 

Fenivessy.     See  Liebermann. 

Fischer,  189. 

Ford,  248,  290,  291,  294. 

Fraenkel  and  Sobernheim,  46,  79,  88, 

134,  168,  206,  304. 
Frasey,  221. 
Freundlich,  424. 
Friedberger.     See  Pfeiffer. 
Friedemann.     See  Neisser. 
Friedemann,  U.,  516. 
Frouin,  510,  524. 

Gaidukow.     See  Englemann. 

Gameleia,  1. 

Garbowski,  415. 

Gay,    345,    366,    367,    442,    458,   509, 

512.     See  also  Bordet. 
Gengou,  346,  347,  348,  356,  358,  398, 

403,  404,  431,  442,  449,  458,   500, 

510,  512,  514,  516,  517,  524.     See 

also  Bordet. 
Girard-Mangin    and    Henri,  313,  314, 

316,  318,  319,  323,  330,  442,  431,  511. 
Gley.     See  Camus. 
Grassberger   and    Schattenfroh,    519, 

520,  521,  526,  528. 
Gratia  and  Lienaux,  492. 
Gruber,  81,  85,  90,  91,  97,  101,  102, 

143,  144,  146,  148,  149,  150. 
Gruber  and  Durham,  93,  95,  118,  136, 

505. 

Griinbaum,  253. 
Guthier,  415. 

Hahn  and  Tromsdorff,  518. 
Hamburger,  415. 
Hamburger,  F.,  251,  253,  256. 
Hammersten,  25n 
Hankin,  24,  25. 
Hardy,  323,  419 
Heinrich,  415. 


Henri.     See  Girard-Mangin. 

Henri,  Lalou,  Mayer  and  Stodel,  313, 

330,  516. 

Henri  and  Mayer,  328,  416. 
Henzel.     See  Czaplewski. 
Hiine.     See  Neufeld. 

Inmann.     See  Levaditi. 

Issaef,  33,  48,  52,  88,  100,  120.     See 

also  Pfeiffer. 
Ivanhoff,  88. 

Jagic.     See  Landsteiner. 

Jochmann  and  Krause,  475,  476,  483. 

de  Jordis,  426. 

Klein,  383,  399,  453. 

Klemperer,  470. 

Koessler.     See  Levaditi. 

Kolle,  223. 

Kossel,  176,  197,  281. 

Kraus,  145,  146,  148,  159,  362. 

Kraus  and  Eisenberg,  304,  308. 

Kraus  and  Lipschiitz,  283. 

Kraus  and  v.  Pirquet,  500. 

Kraus  and  Seng,  150. 

Krause.     See  Jochmann. 

Kyes,  513. 

Kyes  and  Sachs,  367. 

Lalou.     See  Henri. 
Lambotte,  479. 
Landsteiner,  211,  384,  518. 
Landsteiner  and  Jagic,  312,  314,  431, 

442,  500,  516,  520,  523,  525. 
Landsteiner  and  Reich,  498. 
Lea,  415. 
Leblanc,  251. 
Leclainche  and  Valle*,  256. 
Leclef.     See  Denys. 
Leishmann,  389. 
Lemoine.     See  Linossier. 
Lesourd,  479. 
Leuriaux,  478. 
Levaditi,  397. 

Levaditi,  Inmann  and  Koessler,  390. 
Liebermann  and  Fenivessy,  399. 
Lienaux.     See  Gratia. 


INDEX  OF  AUTHORITIES. 


533 


Linossier  and  Lemoine,  253. 
Lipschiitz.     See  Kraus. 
Lipstein,  357. 
Lipski,  10. 
Lottermoser,  415,  426. 

Madsen,  283,  518.  See  also  Arrhenius, 
Dyer. 

Malfitano,  425. 

Malvoz,  158,  244,  432. 

Manicatide,  478. 

Marchand.     See  Denys. 

Marmorek,  13,  104,  112,  116,  117. 

Martin  and  Cherry,  204,  259. 

Martin.     See  Muir. 

Massart,  76,  103. 

Massart  and  Charles  Bordet,  3,  11,  12. 

Mayer.     See  Henri. 

Mertens,  256. 

Mesnil,  10,  35,  98. 

Metchnikoff,  1,  2,  7,  9,  18,  19,  24,  25, 
32,  34,  51,  57,  68,  74,  79,  81,  83 
85,  86,  88,  90,  98,  100,  103,  105, 
120,  130,  134,  140,  162,  186,  197, 
206,  207,  208,  211,  241,  242,  307, 
389,  396,  503,  526,  528,  529. 

Meyer,  524. 

Michaelis,  500. 

Mijers,  246,  251. 

Moreschi,  366,  405,  407. 

Morgenroth,  261,  274,  275,  297,  298, 
299,  300,  301,  355,  358,  518.  See 
also  Ehrlich. 

Morgenroth  and  Sachs,  278. 

Moro,  253. 

Much  and  Liebert,  516. 

Muir,  367,  511,  518. 

Muir  and  Browning,  334,  399,  400, 
402,  447,  509,  510. 

Muir  and  Martin,  390. 

Muller,  415. 

Muller,  399,  522. 

Nasse,  437 

Neisser,  238,  240. 

Neisser  and  Friedemann,  320,  323,  499, 

500,  513,  516,  517. 
Neisser  and  Wechsberg,  299,  356,  357, 

362,  366. 


Nernst,  516,  523. 
Neufeld  and  Hiine,  390. 
Neufeld  and  Rimpau,  390. 
Neufeld  and  Topfer,  396. 
Nicolle,  145,  146,  148. 
Noguchi,  367. 
Nolf,  251. 
Nuttall,  253. 

Obermeyer  and  Pick,  525. 
Otto  and  Sachs,  518,  523. 

Paal  and  Amberger,  415. 

Paal  and  Voos,  415. 

Paltauf,  147,  148. 

Pasquale,  27. 

Pauli,  422,  516,  519. 

Perrin,  312. 

Petruschy,  117. 

Pfeiffer,  19,  27,  34,  43,  45,  56,  57,  58, 
60,  63,  77,  78,  81,  82,  83,  85,  86,  87, 
91,  94,  95,  97,  102,  134,  136,  137, 
149,  161,  206,  207,  208,  212,  213, 
214,  223,  241,  475,  477,  483,  503,  504. 

Pfeiffer  and  Friedberger,  283,  285, 
294,  301,  304,  305,  351,  359,  399, 
407,  408,  409,  412. 

Pfeiffer  and  Issaef,  59. 

Pick.     See  Obermeyer. 

Porges,  500,  517. 

Quincke,  420,  421,  422. 

Remy,  406. 

Rimpau.     See  Neufeld. 

Roger,  130,  146.     See  also  Charrin. 

Rondini.      See  Sachs. 

Rothland,  415. 

Roux,  79. 

Roux  and  Vaillard,  277,  278,  296. 

Sabatini,  435. 

Sachs,   340,   345,  351,  352,  353,  359, 

399,  400,  407,  408,  409,  410,  412,  515, 

521,  523.      See  also  Ehrlich,   Kyes, 

Morgenroth  and  Otto. 
Sachs   and  Bauer,  443,  446,  447,  448, 

449,  450,  451.  452,  456,  459,  460, 

461   511. 


534 


INDEX  OF   AUTHORITIES. 


Sach  and  Rondini,  460 
Salimbini,  100. 
Samoiloff,  10. 
Sanarelli,  79,  120. 
Sauerbeck,  389. 
Savtchenko,  1,81,  389,  395. 
Schutze,  256,304.  See  also  Wassermann. 
Seng.     See  Kraus. 
Shattenfroh.     See  Grassberger. 
Sleeswijk.      See  Bordet. 
Sobernheim.     See  Fraenkel. 
Sollbildner,  426. 
Spengler,  476. 
Spiro,  425. 
Spring,  415. 

van  Steenberghe,  251,  469. 
Stern,  253. 
Stodel.     See  Henri. 
Streng,  448,  507,  511,  515.     See  also 
Bordet. 

Tchistowitch,  148,  175,  246,  249,  255, 

257. 
Topfer.     See  Neufeld. 


Toyosumi,  524, 
Tromsdorff.      See  Hahn. 
Trumpp,  149,  150. 

Vaillard.     See  Roux. 
Valle".     See  Leclainche. 
Vincenzi,  478. 
Volk.     See  Eisenberg. 
Voos.     See  Paal. 

Wassermann,  189,  226,  246,  281,  290, 

291,  294. 

Wassermann  and  Bruck,  468,  479. 
Wassermann  and  Schiitze,  253. 
Wechsberg.     See  Neisser. 
Weil,  500. 
Werigo,  11,  12,  13. 
Widal,  222. 
Woronin,  11,  12. 
Wright  and  Douglas,  390. 

Zaugger,  516. 
Zuelzer,  256. 


INDEX  OF   SUBJECTS. 


NOTE. — The  numbers  printed  in  BOLD-FACED  type  refer  to  pages  on  which  the  topic  is  specifically 

discussed.  « 


Absorption  — 

compared  with  adsorption,  415. 

in  variable  proportions,  263. 

Alexin,    and   the   antagonistic   prop- 
erty of  normal  serum,  398. 

(see  also  Fixation  Reaction). 
Absorption  Experiments  — 

as  producing    condition    of    equilib- 
rium, 238. 

showing  multiplicity  of  normal  ag- 
glutinins,  161. 

Cause  of  error  in,  383,  509. 

Technic  of,  171. 
Adaptability  — 

of  bacterial  cultures  in  the  body  of 
immunized  animals,  1. 

Failure  of,  of  cultures  in  vitro,  75. 
Adsorption  — 

between  cells  and  chemical  precipi- 
tates, 431. 

between  non-cellular  substances,  414 
et  seq,  514. 

compared  with  absorption,  415. 

Relation  of,  to  concentration,  418. 

The  phenomena  of,  414,  516  et  seq. 

The  phenomena  of,  and  the  conglu- 
tinin  of  bovine  serum,  440. 

(see  also  Molecular  Adhesion"). 
Agar,  compared  with  gelatine  as  a  cul- 
ture medium   23,  29. 
Agglutination  — 

as  a  phenomenon  of  molecular  adhe- 
sion, 93, 144, 154, 158, 163,419,517. 

as  cause  of  error  in  bacteriolytic  ex- 
periments, 152. 

compared     with     coagulation,     153, 
157,  163,  182   506. 

compared     with     dissociation,     325, 
432. 

compared  with  fixation  reaction,  486. 

of  bacteria,  65,  88,  90,  96,  99,  100, 
134,  504. 

(see  also    under   each   micro-organism 
and  under  Antimicrobial  Sera). 

of   casein    particles   by   lacto-serum, 
150,  155. 

of  clay  suspensions  by  NaCl,  153. 

of  killed  bacteria,  93. 

of    red    blood    corpuscles,    134,    165, 
312      (see  also  H emagglutination) . 


Agglutination  —  Continued. 

Accuracy  of,  as  a  means  of  diagnosis 
of  bacteria,  96. 

Artificial  resistance  of  bacteria  to,  98. 

Division  of,  into  two  phases,  158,  163. 

Dissociation    of,    by    chemical    pre- 
cipitates, 313,  318,  322,  324. 

Effect  of,  on  morphology  of  bacteria, 
91,  92,  93,  149. 

Effect  of  endorcorpuscular  salts  on, 
313,  314,  316. 

Effect  of  heat  on,  93. 

Effect  of  mechanical  motion  on,  150. 

Effect  of  salt  on,  151. 

Inhibition   of,   by  serum,   313,   318, 
321,  485. 

Inhibition  of,  by  sodium  citrate,  432. 

Mechanism  of,  142. 

Relation  of,  to  immunity,  99,  102. 

Relation  of,  to  motility  of  bacteria, 
91,  143,  147,  183. 

Theories  of,  91,    143,  145,  147,  149, 

163,  499. 
Agglutinins  — 

compared  with  conglutinins,  448. 

compared  with  precipitins,  149. 

for  bacteria,  149. 

for  red  blood  corpuscles  (see  Hemag- 
glutinins). 

in  general,  91,  500. 

Absorption  of,  by  cells,  161,  262,  520. 

Comparison    of,    in  normal  and  im- 
mune sera,  181,  290. 

Effect  of  heat  on,  96,  181. 

Identity  of  normal  and  immune,  290. 

Multiplicity    of,    in    normal    serum, 
161,  240. 

Relation  of,  to  opsonins,  395. 

Relation  of,  to  precipitins,  149. 

Relation  of,  to  preventive  substance, 
97. 

Relation   of   molecular   adhesion  to, 
158,  163,  419. 

Specificity  of,  97. 

Variations  in,  to  a  given  micro-organ- 
ism, 527. 
Albuminous  Substances  — 

Sensitizers  of  sera  active  against,  241, 
246,  257,  301. 

Specificity  of  sensitizers  for,  253. 


535 


536 


INDEX   OF  SUBJECTS. 


Alexin  (syn.  Bactericidal  Property;  Bac- 
tericidal Substance;  Complement 
(Ehrlich),  q.  v.)  — 

absorption  and  the  antagonistic 
power  of  serum,  398. 

in  normal  serum,  89,  101,  135,  170. 

Action  of,  149,  161,  174,  189. 

Action  of,  on  its  proper  corpuscles, 
173,  187,  198,  333. 

Antitoxin  for  (see  Anti-alexin}. 

Comparison  of  combining  and  toxic 
properties  of,  340. 

Death  of  infection  not  due  to  lack 
of,  226. 

Effect  of  decalcification  on,  435. 

Ehrlich's  phenomenon  in  union  of, 
with  anti-alexin,  268,  271. 

Evidence  for  multiplicity  of,  237,  238. 

Justification  of  term,  187. 

Mode  of  union  with  anti-alexin,  268. 

Pfeiffer's  conception  of,  213. 

Reactivation  of  heated  serum  by, 
137,  138. 

Relation  of,  to  opsonins,  390. 

Relation  of,  to  sensitizing  substance, 

172,  214,  229,  234,  239,  334,  357, 
363,  368,  387,  403,  440,  511. 

Union  of,  with  sensitized  cells,  196, 

261,  337,  365. 
Unity  of,  in  a  given  serum,  140,  189, 

211,216, 219, 328, 235, 382, 405, 507. 
Variations  in,  from  different  animals, 

173,  187,  198,  333,  507. 

Alexin  Deviation  (syn.  N  eisser-W  echs- 
berg  Phenomenon;  Complement  De- 
viation) — 

due  to  serum  precipitates,  355,  358. 

in  hemolysis,  299,  357. 

Incorrect  explanations  of,  352,  366. 
Alexin  Fixation  — 

by  serum  precipitates,  346. 

on  sensitized  cells,  190,  219,  230,  234, 
244,  261,  363,  365,  398,  510. 

Antagonistic  effect  of  normal  serum 
on,  351,  352,  398,  402,  410,  509. 

Effect  of  sodium  citrate  on,  404,  512. 

Inhibition  of,  by  antisensitzer,  287. 

Relation  of  normal  amboceptors  to, 
399. 

(see  also  Fixation  Reaction;  Molecular 

Adhesion). 
Alum  — 

Agglutination  of  bacteria  by,  432. 
Amboceptor    (Ehrlich   and    Morgenroth 
syn.  for  Sensitizing  Substance),  230, 
233,  340,  363,  388,  458,  459,  510. 

Normal,  as  cause  of  non-fixation  of 

alexin,  399. 
Amphophiles  — 

in  peritoneal  exudate,  33,  54. 

Importance  of,  in  phagocytosis,  10. 


Antagonistic  Property  of  Normal  Serum, 

see  Normal  Serum. 

Anthrax  Bacillus,  see  Bacillus  Anthracis. 
Anti-Agglutinins  — 

in  antihematic  sera,  176,  205. 
Reaction    of    normal    and    immune 

agglutinins  to,  290. 
Anti-Alexin,  198,  448. 

Action  of,  on  alexin,  203,  204,  205, 

216,  268,  519. 
Antihemolytic    and    antibactericidal 

effect  of,  201,  216. 
Effect  of  heat  on,  199. 
Ehrlich's    phenomenon    in    mixtures 

of,  and  alexin,  268,  271. 
Equilibrium  between,  and  alexin,  274. 
Specificity  of,  200,  202,  216. 
Anti-Antibodies,  280,  306  (see  Antisen- 

sitizers;   A  nti-agglutinins  ) . 
Antibactericidal   Effect,  see  under  Anti- 
alexin;   Antihemotoxic    Serum;   An- 

tih/tic  Properties  of  Normal  Serum. 
Antibodies  — 

Classification  of,  501. 

Relation  of,  to  receptors,  296,   301, 

305,  497. 
Relation  of  origin  of,  to  their  action, 

280. 
Union  of,  with  antigen,  440,  501,  506. 

515,  523. 
(see     Preventive    Substance;  Immune 

Bodies;  Sensitizing  Substance;  Pre- 

cipitins;  Agglutinins;  Amboceptors). 
Anticholera  Serum  (as  type  of  Bacteri- 

olytic  Sera). 
Action  of,  on  cholera  vibrio,  22,  29, 

44,  47,  65,  173. 

Agglutination  of  vibrios  by,  65, 97, 103. 
Analogies    between,    and    hemolytic 

sera,  140. 

Diagnosis  of  vibrios  by,  60,  94. 
Effect  of,  on  leucocytes,  51  et  seq. 
Effect  of,  on  chemiotaxis,  52,  80. 
Effect  of  heat  and  conservation  on, 

47,  58,  59,  65,  66,  69. 
Formation    of    specific    precipitates 

with,  145. 

Preventive  substance  in,  51  et  seq. 
Specificity  of,  94,  97. 
(see  also    V.  cholerce;   Pfeiffer's   Phe- 
nomenon). 
Anticorpuscular    Serum    (syn.    Hemoly- 

sin;  Hemolytic  Serum;  Hemotoxin). 
Antigens,  250,  523. 

Accessory  nature  of,  526  et  seq. 
Union  of,  with  antibodies,  440,  501, 

506,  515,  523. 
Antihematic     Serum     (syn.     Hemolytic 

Serum,  q.  v.) 
Antitoxic  properties  of ,  175,  185,  186, 

197  (see  also  Antihemotoxic  Serum). 


INDEX  OF    SUBJECTS. 


537 


Antihematic  Serum  —  Continued. 

Effect  of,  on  blood  corpuscles,   167. 
Fixation  of  properties  of,  by  specific 

red  blood  corpuscles,  171. 
Properties  of,  166. 
Antihemolytic  Serum,   281   (syn.   Anti- 

hemotoxic  Serum,  q.  v.). 
Antihemotoxic  Serum,  186,  197. 

Dual  nature  of,  198,  281   (see  Anti- 

sensit  izer;  A  nti-alexin ) . 
Antihemolytic    and    antibactericidal 

nature  of,  201,  281. 
(see    also    under     Hemolytic    Serum; 

Antihematic  Serum}. 
Antihuman  Serum,  Reactions   of,  with 

human  products,  256. 
Antilytic  Properties  of  Normal  Serum, 

see  Normal  Serum. 
Antimicrobial  Sera  — 

On  the  existence  of  sensitizing  sub- 
stances in  the  majority  of,  217. 
(see    also   Anticholera    Serum;   Anti- 
streptococcus    Serum ;    also    under 

each  Micro-organism}. 
Antisensitizers,  198. 

considered  as  solution  of  receptors, 

297. 

to  normal  sensitizers,  285. 
Action  of,  on  sensitizer,  286,  287,  513. 
Conception  of,  by  Ehrlich  and  Mor- 

genroth,  301. 
Effect  of  normal  serum  on  action  of, 

288,  293. 
Effect     of     unfavorable     media     on 

action  of,  296. 

Inhibition  of  alexin  fixation  by,  287. 
Preventive   and   curative   action   of, 

283. 

Properties  of,  280,  283. 
Unity  of,   active   against   sensitizers 

from  a  given  animal  species,  290. 
Antistreptococcus  Serum  — 

Agglutination  of  streptococci  by,  118. 
Contribution  to  the  study  of,  104. 
Effect   of,   on   streptococci   in   vitro, 

117. 
Effect   of,    on   streptococci   in    vivo, 

121,  131. 

Preventive  action  of,  116. 
Relation  of,  to  delayed  phagocytosis, 

127. 

(see  also  Streptococcus'). 
Antitoxin  — 

Action  of,  on  toxin,  204,  259,  295, 518. 
Ehrlich 's    phenomenon    with    toxin, 

mixtures,  264. 

Natureof  toxin,  union,  260etseq., 267. 
Origin  of,  from  toxin  (Buchner),  51. 
Union  of,  with  toxon,  267. 
(see      also      Diphtheria      Antitoxin; 

Toxins). 


Antituberculous    Sensitizers,    see   under 

Bacillus  Tuberculosis. 
Aqueous  Humor  — 

in  vaccinated  animals,  73. 

Absence  of  preventive  substance  in, 

73,  289. 

Anthrax  bacillus  in,  7,  26. 
Bactericidal  property  of,  26,  29,  71. 
Areola  — 

formed  about  resistant  streptococci, 

106,  109. 
Relation  of,  to  negative  chemiotaxis, 

113. 

Relation  of,  to  virulence,   113. 
Attenuation  of  Bacteria,  3. 
Avian  Diphtheria,  492. 

Bacteriology  of,  492,  493. 

Pathogenesis  of,  494. 

Relation   of,    to    human    diphtheria, 

492. 

Avian  Tuberculosis,  see  Bacillus  Tuber- 
culosis. 

Bacillus  Anthracis  — 

in  the  aqueous  humor,  7,  26. 

Antiserum  for,  221. 

Effect  of  heated  rat  serum  on,  181. 

Effect  of  rabbit  serum  on,  26. 

Opsonins  for,  391  et  seq. 

Phagocytosis  of,  35,  36. 

Phagocytosis  of,  in  relation  to  opso- 
nins,  39C. 

Sensitizers  for,  in  antiserum  to,  221. 
Bacillus  Coli— 

Agglutination  of,  91. 

Phagocytosis  of,  35. 

Bacillus  of  Danysz,  Phagocytosis  of,  35. 
Bacillus  Diphtheria,  10. 

Phagocytosis  of,  35. 
Bacillus  of  Hog  Cholera  — 

Antiserum  against,  52. 

Phagocytosis  of,  35. 
Bacillus  Influenzae  — 

Etiological  relation  of,  to  whooping 
cough,  475. 

Reactions  of,  with  sera  of  whooping- 
cough  patients,  480. 

Bacillus  Muscoides,  Phagocytosis  of,  36. 
Bacillus  Pestis,  Sensitizers  for,  in  anti- 
serum  to,  219. 
Bacillus  Proteus  — 

Phagocytosis  of,  14,  36. 

Pfeiffer's  phenomenon  with,  224. 

Sensitizers  for,  in  antiserum  to,  223. 
Bacillus  Pyocyaneus,   Phagocytosis    of, 

37. 
Bacillus  of  Swine  Plague,  Sensitizers  for, 

in  antiserum  to,  221. 
Bacillus  Typhosus  — 

Agglutination  of,  91,  181,  500. 

Diagnosis,  of,  95. 


538 


INDEX    OF    SUBJECTS. 


Bacillus  Typhosus  —  Continued. 

Opsonin  for,  393. 

Phagocytosis  of,  35. 

Sensitizers  for,  in  antiserum  to,  221. 
Bacillus  Tetani,  10. 

Agglutination  of,  91,  92,  99. 
Bacillus  Tuberculosis  — 

Action  of  various  mammalian  types 
of,  in  producing  sensitizers,  464. 

Relation    between    types   of,    as    re- 
gards sensitizers,  467. 

Sensitizers  for,  462,  464,  467. 
Bacillus  Vermicularis  — 

Phagocytosis  of,  36. 

Bacillus   of   Whooping-cough     (Bordet- 
Gengou),  473,  482,  488. 

Action    of    antiserum    to,    483,    485. 
487. 

Antigens  of,  526. 

Comparison    of,   with   B.    influenzse, 
483. 

Cultural  characteristics  of,  478,  482, 
484. 

Endotoxin  of,  487,  488,  490. 

Isolation  of,  474,  477,  482. 

Morphology  of,  474,  478,  484. 

Pathogenicity  of,  478,  486. 

Reactions  of,  with  sera  of  whooping- 
cough  patients,  479,  482. 

Stain  for,  478. 
Bacteria  — 

Action  of  preventive  substance  6n, 
90,  225. 

Adaptation    of,    in    immunized    ani- 
mals, 1. 

Agglutination  of,  65,  88,  90,  134. 
(see  also  under  each  micro-organism). 

Agglutination  of,  by  alum,  432. 

Agglutinins  for,  149. 

Artificial  resistance  of,  to  agglutina- 
tion, 98. 

Attenuation  of,  3. 

Chemiotaxis  by,  3,  4,  11. 

Diagnosis  of,  by  agglutination,  96. 

Increase  in  pathogenicity  of,  2,  6. 

Modified  cultures  of,  2  et  seq. 

Selection  of,  by  phagocytes,  6,  16. 
Bactericidal  Immunity,  48. 

Hankin's  theory  of,  24. 

Denys'  theory  of,  25. 
Bactericidal  Properties   (of  body  fluids), 
17  et  seq.,  20. 

in  immune  serum,  42,  46,  50,  68,  70. 

in  normal  serum,  26,  45,  46,  68,  69, 
70,  75,  78,  201. 

in  relation  to  the  number  of  leuco- 
cytes, 24,  30,  32,  84. 

in  serum  compared  with  peritoneal 
exudate  and  with  plasma,  21,  27. 

of  aqueous  humor,  26,  29,  71. 

of  edema  fluid,  27,  29,  31,  71. 


Bactericidal  Properties  —  Continued. 
of  phagocytes,  24,  33,  36. 
of  plasma,  27. 
of  serum    as   due   to    cooperation   of 

two  substances,  89,  100,  135. 
Absence  of,  in  intestinal  transudate, 

74. 
Comparison  of  potency  of,  of  normal 

and  immune  sera,  45,  69,   75,  78, 

177,  201. 

Effect  of  heat  on,  67,  72. 
Effect  of  infections  on,  67,  72. 
Effect  of  injections  of  normal  serum 

or  bouillon  on,  31,  32,  67,  87. 
Method  of  determining,  of  a  fluid,  20. 
Non-specificity  of,   in  immune  sera, 

42,  46,  50,  68. 
(see      also      Bactericidal      Substance; 

Alexin). 

Bactericial  Substances  (of  body  fluids)  — 
Absence  of,  in  edema  fluid,  71,  72. 
Absence  of,  in  intestinal  transudate, 

73. 

Localization  of,  during  life,  82. 
Origin  of,  of  serum,  24,  30,  32,  41, 

68,  84,  90. 
Unity    of,    in    normal    and    immune 

sera,  45,  69,  75,  78,  201. 

(see    also    Bactericidal    Properties; 

Alexin). 

Bacteriolysis,  see  Pfeiffer's Phenomenon. 
Bacteriolytic  Experiments,Cause  of  error 

in,  152. 

(see  also  Pfeiffer's  Phenomenon). 
Bacteriotropins,  syn.  Opsonins. 
Barium  Sulphate  — 

Agglutination  and  hemolysis  of  blood 

by,  313,  315. 
Complexes  of,  with  colloids,  420,  421 , 

500. 
Complexes  of,   with  sodium  citrate, 

425. 
Dissemination  of,  by  sodium  citrate, 

424. 
Dissociating    effect    of    colloids    on, 

416,  419. 
Bordet's  Theory  of  Immunity,  208,  215, 

442,  496  et  seq. 
Bordet's  Theory  of  Agglutination,   143, 

163. 
Bordet's     Theory    of     Toxin-Antitoxin 

Union,  266,  439,  519. 
Body  Fluids  — 

Bactericidal  Theory  of,  17  et  seq. 
(syn.  Humoral  Theory  of  Im nninity). 
Bouillon  — 

Effect   of   injections   of,   on   bacteri- 
cidal property  of  blood,  31,  32,  67, 

87. 

Effect   of   injections   of,   on   strepto- 
coccus infection,  107,  112,  122. 


INDEX     OF    SUBJECTS. 


539 


Bovine  Serum  — 

Hemolysis   of   guinea-pig   blood   by, 

368  et  seq.,  443  et  seq. 
Hemagglutination  by,  370. 
Conglutinin  of,  see  Conglutinin. 

Calcium  Chloride,  Antagonism  to  action 

of,  by  sodium  citrate,  425,  434. 
Calcium  Fluoride  — 

Action  of    sodium    citrate    on,    427, 

432. 

Agglutination  and  hemolysis  of  red 
blood  corpuscles  by,  313,  315,  432. 
Flocculation  of,  by  electrolytes,  315. 
Chemical  Precipitates,  see  under  Precip- 
itates. 
Chemiotaxis  — 

by  bacteria  in  vivo,  3,  4,  11. 

by  bacterial  products,  52. 

by  preventive  serum,  52,  80. 

Effect  of  anticholera  serum  on,  52, 

80. 

Effect  of,  on  phagocytosis,  11  et  seq. 
Negative,  4,  5,  11  et  seq.,  13. 
Negative,    by    streptococci,    13,    15, 

106,  112,  113. 

Relation  of  negative,  by  streptococ- 
cus to  an  areola,  113. 
Chloroform,  Effect  of,  on  phagocytosis, 

23. 

Cholera  Vibrio,  see  Vibrio  Choleras. 
Cholera  Infection,  48. 
in  rodents,  74. 
Course  of,  48. 
Phagocytosis  in,  9,  19,  24. 
(see  also  under  Vibrio  Choleras). 
Clay,  Agglutination  of  suspensions  of, 

153. 

Coagulin,  154,  syn.  Precipitin. 
Coagulation  — 

as   a  phenomenon  of  molecular  ad- 
hesion, 153. 
compared    with    agglutination,    153, 

157,  163,  182,  506. 
Colloids  — 

Action  of,   compared  with    hemoly- 

sins,  404,  517. 
Complexes  of,  with  barium  sulphate, 

420,  421,  500. 
Definition  of,  414. 

Dissociating  effect  of  serum  due  to, 

324. 

Dissociation  of  suspensions  with,  419. 
Flocculation  of,  by  electrolytes,  313, 

421,  422. 

Protection  of  unstable,  by  stable,  330. 
Suspension   of   chemical   precipitates 

by,  313. 

Colloidal  Substance  of  Bovine  Serum 
(Bordet  and  Gay},  syn.  Conglu- 
tinin. 


'Colon  Bacillus,  see  Bacillus  Coli. 
Complement,  syn.  (Ehrlich  and  Morgen- 

roth)  for  Alexin,  233,  299,  363. 
Suitable,  367,  388. 
(see  also   under   Ehrlich's    Theory  of 

Immunity). 
Complementoids  (Ehrlich   and   Morgen- 

roth),  402. 

On  the  existence  of,  333,  340,  512. 
Gay's  interpretation  of,  341,  345. 
Nature  of,  according  to  Ehrlich,  340. 
(see  also   under   Ehrlich's    Theory  of 

Immunity). 
Conglutination,  447. 

as  a  phenomenon  of  molecular  ad- 
hesion, 376,  379. 
Examples  of,  456. 
Necessity  of  sensitizer  and  alexin  in, 

370,  374  et  seq. 
Relation  of,  to  increased  hemolysis, 

456. 
Conglutinin  of  Bovine  Serum,  376,  387, 

440,  444,  512  (Bordet  and  Streng), 

syn.   Colloidal  Substance  of  Bordet 

and  Gay. 

compared  with  agglutinin,  448. 
Effect  of,  on  goat  corpuscles,  457. 
Effect  of,  on  sensitized   and    alexin- 

ized  red  blood  cells,  373  et  seq.,  444. 
Effect  of  dialysis  on,  460. 
Mode  of  action  of,  456. 
Potency  of,  381,  450. 
Proofs  of  accuracy  of  conception  of, 

380  et  seq.,  447  et  seq. 
Culture  Media  — 

for  bacillus  of  whooping-cough,  474. 
for  streptococcus,  104. 
(see  under  Agar;   Gelatine). 
Cytolytic  Sera  — 
Theories  of,  186. 
Mode  of  action  of,  228. 

Denys'  Theory  of  Immunity,  25. 

Dineur's  Theory  of  Agglutination,  147. 

Diphtheria  Antitoxin,  Effect  of,  on  leu- 
cocytes, 10,  37,  77. 

Diphtheria  Bacillus,  see  Bacillus   Diph- 
therice. 

Diphtheria  of   Fowls,  see  Avian  Diph- 
theria. 

Diphtheria  Toxin,  effect  of,  on  phagocy- 
tosis, 37. 

Dissociation  — 

of  agglutination  caused  by  chemical 
precipitates   by   serum,    313,    318, 
322,  324. 
of  suspensions  with  colloids,  419. 

Dissolution   of   Red    Blood    Corpuscles, 
134,  165;    (see  Hemolysis). 

Dog  Red  Blood  Corpuscles,  Fragility  of, 
179. 


540 


INDEX     OF    SUBJECTS. 


Dog  Serum  — 

Antiserum   to,    248,    249,    251,    255, 
258. 

"  Complementoids  "  in,  340. 

Effect  of  heat  on,  340,  345. 

Normal  hemagglutinins  in,  178. 

Normal  hemolysins  in,  179,  340. 

Precipitin  for,  249. 

Dyeing  Phenomena,  Fixation  of  sensi- 
tizing substances  regarded  as,  194, 
216,  230,  262. 

Edema  — 

in  vaccinated  animals,  73. 

Absence  of  bactericidal  substance  in, 

27,  29,  31,  71,  72. 
Means  of  producing,  27,  29. 
Preventive  substance  in,  38  et  seq., 

73. 

Eel  Serum,  Hemolysis  by,  437. 
Egg  Albumin  — 
Antiserum  to,  248. 
Precipitins  for,  248. 
Sensitizers  to,  in  antiserum  to,  248, 

254. 
Ehrlich's    Phenomenon    (of    union    of 

toxin  with  antitoxin},  264. 
in  union  of  alexin  with  anti-alexin, 

268,  271. 

Ehrlich's  Theory  of  Immunity,  294, 
296,  305,  333,  352,  363,  388,  410, 
442,  459,  497,  523. 

(see  also  under   Amboceptors;     Com- 
plements;  Complementoids;  Recep- 
tors.) 
Electrolytes  — 

Effect    of,    on    agglutination    of    red 
blood  corpuscles  by  chemical  pre- 
cipitates, 314  et  seq.,  321. 
Flocculation  of  colloids  by,  313,  421, 

422. 
Endothclium,  Function  of,  in  Pfeiffer's 

phenomenon,  57. 
Endotoxin  — 

of  whooping-cough  bacillus,  488. 
Pathogenicity     of     whooping-cough, 

489. 

Preparation  of,  488,  489. 
Eosinophiles,  10. 

Origin  of  bactericidal  substance  in, 

24. 
Exudate,  see  Peritoneal  Exudate. 

Fibrinogen  — 

Antiserum  to,  249,  250. 
Precipitins  for,  255. 
Sensitizers  in  antiserum  to,  249,  255. 
Fixation  Reaction  (Bordet-Gengou),  syn. 
Reaction  of  alexin  fixation,  189,  243, 
398. 
with  lactoserum,  251,  253. 


Fixation  Reaction  —  Continued. 

Applicability   of,   to   diagnosis,   407 

462,  464,  467,  479,  486,   508. 
Comparison   of,    with   agglutination 

486. 
Comparison    of,    with    precipitation 

249,  252,  255,  256. 
Criticism  of  deduction  drawn  from, 

245,  405. 
Demonstration  of  Sensitizers  by,  218, 

245,  250,  348,  467. 
Specificity  of,  222,  253. 
Technic  of,  219,  247,  404  et  seq.,  469. 
(see  also  under  Alexin  Fixation). 
Flocculation    of   .Colloidal    Complexes, 

313,  421,  422. 
Frog  Serum,  as  alexin  and  opsonin,  391 

et  seq. 

Gelatin,  compared  with  agar  as  culture 

medium  for  vibrios,  23,  29. 
Gels,  414  et  seq. 
Goat   Red   Blood   Corpuscles,   Effect  of 

conglutinin  on,  457. 
Goat  Serum,  Hemagglutination  by,  92, 

178. 

Gruber's  Diagnosis  by  Agglutination,  95. 
Gruber's   Theory  of   Agglutination,  91, 

143,  149. 
Guinea-pig  Serum  — 

Action  of,  on  bacteria,  96. 

Action  of,  on  alien  blood  corpuscles, 

178. 

Normal  hemolysins  in,  179. 
Potency  of  alexin  in,  188. 

Hankin's  Theory  of  Immunity,  24. 
Hemagglutination  (Agglutination  of  red 

blood  cells),  65. 

by  chemical  precipitates,  312,  431. 
by  products  of  cholera  vibrio,  92. 
Effect  of  electrolytes  on,  by  chemical 

precipitates,  314,  321. 
Effect  of,  on  morphology  of  red  blood 

cells,  93. 

(see Hemagglutinins;  Conglutination). 
Hemagglutinins,  370. 
Artificial,  137. 
Effect  of  dilution  on,  174. 
Effect  of  heat  on,  136,  138. 
Normal,     65,     92,     136,     178,     370; 

(hemagglutinins  of  normal  serum). 
Passive  transfer  of,  168. 
Relation  of  normal,   to  hemolysins, 

179. 
(see     Agglutinins;     Anti-agglutinins; 

Hemagglutination). 
Hemolysins,  syn.  Hemoly  tic  Serum  \Anti- 

licinatic  Scrntn;  Anticorpuscular  Se- 
rum;   Hctnotoxin. 
compared  with  colloids,  404,  517. 


INDEX    OF    SUBJECTS. 


541 


Hemolysins  —  Continued. 

Activity  of,  due  to  two  substances,184. 

Analogy    of,  with    bactericidal    sub- 
stance, 140,  167,  177. 

Effect  of  heat  on,  138. 

Mode  of  union  of,  with  cell,  436. 

Normal,  179,  237,  340,  367. 

Reactivation  of  artificial,  by  normal 
serum,  138. 

Specificity  of,  139,  171. 

Toxicity  of,  139. 

(see  Hemolysis,  Hernoly tic  Serum,  etc.). 
Hemolysls,  134,  165,  186,  333. 

by  chemical  precipitates,  313. 

by    normal    serum,    136;  (see    under 
Hemolysins). 

by  specific  serum  in  vivo,  139. 

by  venom,  367,  433,  513. 

Alexin  deviation  in,  299,  357. 

Effect  of  heat  on,  169. 

Inhibition    of,    by    sodium    citrate, 
404,  433. 

Relation  of  conglutination  to,  456. 

Effect  of  salt  solution  in  experiments 
in,   296,   301,   335,   341,   383,  399, 
451,  453,  456. 
Hemolytic  Serum  — 

and  its  antitoxins,  186. 

produced  by  injecting  stromata,  194. 

Antitoxin    to,    197;  (see    Antihemo- 
lytic  Serum). 

Data  concerning,  229. 

Precipitins  in,  148. 

Reactivation  of,   by  normal  serum, 
138. 

Toxicity  of,  197. 

Union  of,   with   corpuscles  in  vary- 
ing proportions,  194,  260,  521. 

(see    also     Hemolysin;     Antihematic 

serum). 

Hemotoxin,  syn.  Hemolytic  serum,   He- 
molysin. 

Hemosensitizer  or  Hemo sensitizing  Sub- 
stance; (see  under  Sensitizing  sub- 
stance for  blood  corpuscles). 
Hen  Serum  — 

Hemagglutinins  in,  136,  178. 

Hemolysins  in,  179. 

Hog    Cholera    Bacillus,  see   under    Ba- 
cillus of. 
Horse  Serum  — 

Hemagglutinins  in,  92,  178,  370. 

Agglutination  of  bacteria  by,  96,  99, 
100. 

Alexin  of,  367,  368,  371,  379,  444. 

Relation  of  opsonin  to  alexin  in,  392. 

Sensitizer  in,  372,  379,  444. 

Immune   Bodies,  syn.  Sensitizing  Sub- 
stances. 
Single  nature  of  hemolytic,  333. 


Immune  Serum,  syn.  Preventive  Serum. 

q.  v. 
compared    with    normal    serum,    45, 

69,  75,  78,  177,  201. 
Action   of,    due   to   two    substances, 

46,  89. 

Agglutinins  of  normal  and,  290. 
Bactericidal  substance  in,  42,  46,  50, 

68,  70. 

Effect  of  heat  on,  46,  59,  66,  69. 
Inhibition  to  agglutination  by  excess 

of,  485. 

Opsonins  of,  396. 
Specificity  of,  525. 
Immunity  — 
Active,  75,  103. 
Bactericidal,  48. 
Mechanism  of,  75. 
Passive,  79,  99,  101,  102,  303,  304. 
Relation  of  agglutination  to,  99,  102. 
Relation  of  leucocytes  to,  9. 
Theories  of,  18,  24,  25,  206,  208,  212, 

380,  294,  303,  366. 
(see    also    under  Ehrlich's   Theory  of 

Immunity  and   Bordet's   Theory  of 

Immunity). 
Immunized    Animals,    Adaptability    of 

bacterial  cultures  in  the  body  of,  1. 
Infections  — 

Death  of,  not  due  to  lack  of  alexin, 

226. 
Effect  of,  on  bactericidal  properties 

of  serum,  67,  72. 
Mixed,  due  to  phagocytic  selection, 

16. 
(see    Cholera   Infection',    Streptococcus 

Infection). 

Intestinal  Transudate,  Absence  of  bac- 
tericidal substance  in,  73. 

Lactic  Acid,  Effect  of,  on  phagocytosis, 

12. 
Lactoserum,  246. 

Action    of,    compared    with    remain, 
155. 

Fixation  reactions  with,  251,  253. 

Flocculation  of  casein  by,  155,  506. 

Precipitins  of,  156,  253. 

Sensitizers  of,  253. 
Leucocytes  — 

of   rabbit   and    guinea-pig   in   strep- 
tococcus infection,  114. 

Artificial  lowering  of,  in  blood,  30. 

Effect  of  anticholera  serum  on,  51. 

Effect  of  bacterial  products  on,  51, 
53,  120. 

Effect    of    diphtheria    antitoxin    on, 
10,  37,  77. 

Origin  of  bactericidal  substances  in, 
24,  32,  75,  78. 


542 


INDEX     OF    SUBJECTS. 


Leucocytes  —  Continued. 

Relation  of,  to  bactericidal  power  of 
serum,  24,  30,  32,  41,  68,  84,  90. 

Relation  of,  to  immunity,  9. 

Relation  of,  to  preventive  power  of 
serum,  38,  41. 

Relation  of,  to  Pfeiffer's  phenome- 
non, 71,  85. 

(see  also  Phagocytes;  Amphophiles; 
Macrophages;  Microphages) . 

Macrophages  — 

in    exudate    caused    by    diphtheria 

antitoxin,  37. 

in  streptococcus  infection,  128,  130. 
Origin  of  granules  in,  38. 
Mastic  — 

as  a  reagent  for  blood  serum,  347. 
Agglutination  of  blood  corpuscles  by, 

432. 
Dissemination  of,  by  sodium  citrate, 

424. 
Microphages,  in  streptococcus  infection, 

14,  128. 

Modified  Cultures,  (of  bacteria),  2  et  seq. 
Molecular  Adhesion,  414. 

Alexin  fixation  as  a  phenomenon  of, 

365,  403,  514. 
of  conglutinin,  376,  379. 
Agglutination  in  relation  to,  93,  144, 

154,  158,  163,  419,  517. 
Coagulation  in  relation  to,  153. 
(see  Absorption;  Adsorption). 

Negative  Chemiotaxis,  see  under  Chemio- 

taxis. 
Neisser-Wechsberg      Phenomenon,    see 

under  Alexin  Deviation. 
Nictitating  Membrane,  as  means  of  puri- 
fying   cultures  of   avian    diphtheria, 

493. 

Nicolle's  Theory  of  Agglutination,  145. 
Normal  Serum  — 

compared  with  immune  serum,    45, 

69,  75,  78,  177,  181,  201,  290. 
Bactericidal  property  in,  26,  45,  46, 

68,  69,  70,  75,  78,  201. 
Action  of,  with  preventive  serum  in 
Pfeiffer's     phenomenon,     58,     66, 
103,  135. 
Agglutination  of  alien  blood  by,  65, 

92,  136,  178,  181,  290,  370. 
Antagonistic     effect    of,     on     alexin 
fixation,  351,  352,  398,  402  et  seq., 
410,  509. 

Alexin  in,  89,  101,  135,  170. 
Action    of,    on    antisensitizers,    288, 

293. 

Effect  of  injection  of,  31,  32,  67,  87. 
Multiplicity   of   agglutinins   in,    161, 
240. 


Normal  Serum  —  Continued. 
Opsonins  of,  390. 
Reactivation  of  hemolytic  serum  by 

138. 
Sensitizers    in,    184,    237,    240,    288 

372,  379,  444. 
Suppression  of  cure  of  antisensitizer 

by,  288. 

Opsonins  — 

of  frog  serum,  391. 

of  immune  sera,  396. 

of  normal  sera,  390. 

Effect   of,    on    sensitized    red    blood 

corpuscles,  395. 
Nature  of,  389. 

Relation  of,  to  agglutinins,  395. 
Relation  of,  to  alexin,  392. 
Relation  of,  to  phagocytosis,  389  et 

seq. 
Relation  of,  to  sensitizers,  397. 

Paltauf's  Theory  of  Agglutination,  147. 
Pathogenicity,  Increase  of,  in  bacteria, 

2,  6. 

Peritoneal  Exudate,  in  infections  with 
Streptococcus,  110,  124;  (see  also 
Streptococcus). 

in  infections  with  V.  Massaouah,  40. 

Bactericidal  power  of,  21. 

Method  of  producing,  33. 

Non-antagonism  of,   to   alexin   fixa- 
tion, 412. 

Stains  for  study  of  phagocytosis  in, 

33. 

Pfeiffer's  Phenomenon  (syn.  Bacterio- 
lysis), 56. 

in  vivo,  57,  83. 

in  vitro,  48,  57,  58,  83. 

with  Vibrio  Cholerse,  58-60. 

with  Vibrio  Metchnikovi,  64. 

Combined  action  of  preventive  and 
normal  serum  in,  58,  66,  103,  135. 

Comparison  of,  in  vitro  and  in  vivo, 
214,  413. 

Diagnosis  of  vibrios  by,  44,  59,  61,  94. 

Factors  concerned  in,  65. 

Pfeiffer's  explanation  of,  57,  85,  95, 
207,  212. 

Relation  of  endothelial  cells  to,  57. 

Relation  of  leucocytes  to,  71,  85. 

Stains  for  demonstrating,  63. 
Pfeiffer's  Theory  of  Immunity,  212,215. 
Phagocytes  — 

Bactericidal  properties  of,  24,  33,  36. 

Effect  of  chemiotaxis  on,  11  et  seq. 

Selection  of  bacteria  by,  4,  6,  16. 

Tactile  reaction  of,  12. 

(see  also  Leucocytes;  Phagocytosis). 
Phagocytic  Crisis  in  Streptococcus  In- 
fection, 124,  127. 


INDEX    OF    SUBJECTS. 


543 


Phagocytlc  Theory    of   Immunity,    18, 

25;  (see  also  under  Immunity). 
Phagocytosis,  9,  10,  22,  24,  528. 

as  produced  essentially   by  ampho- 
philes,  10. 

of  various  bacteria   (see  under    each 
micro-organism). 

in  cholera  infections,  9,  19,  24. 

in  vaccinated  animals,  11. 

in  vitro,  33. 

in  streptococcus  infection,    14,    108, 
128,  130. 

Delayed,  in  streptococcus  infection, 
124,  127. 

Effect  of  chemiotaxis  on,  11  et  seq. 

Effect  of  chloroform  on,  23. 

Effect  of  diphtheria  toxin  on,  37. 

Effect  of  lactic  acid  on,  12. 

Relation  of,  to  immunity,  99,  206. 

Relation  of  opsonins  to,  389  et  seq. 

Stains  for,  22,  33. 
Phagolysis  in    streptococcus    infection, 

105. 

Pigeon  Serum,  Hemagglutinins  in,  178. 
Plague  Bacillus,  see  Bacillus  Pestis. 
Plasma  — 

Bactericidal  properties  of,  27. 

vProduction  of  artificial,  27. 

(cf.  Edema). 

Polymorphonuclear  Leucocytes,  see  Mi- 
cro phages. 

Precipitins    (precipitating  serum),   175, 
184,  205,  246,  251,  347,  522. 

for  albuminous  derivatives,  251. 

for  dog  serum,  249. 

for  egg  white,  248. 

for  fibrinogen,  255. 

in  lactoserum,  156,  253. 

for  rabbit  serum,  249. 

Comparison  of,  with  agglutinins,  149. 

Comparison  of  reaction  of,  with  coag- 
ulation, 157. 

Comparison  of  reaction  of,  with  fixa- 
tion reaction,  249,  252,  255,  256. 

Effect  of  heat  on,  157. 

Specificity  of,  253,  255. 

Technic  of  reaction  of,  175,  349. 
Preclpltinogen  (precipitable  substance)  — 

in  insufficiently  washed  blood,  347. 

Effect  of  heat  on,  157. 
Precipitates  — 

Chemical  precipitates: 

Hemagglutination  by,  312,  431. 
Hemolysis  by,  313. 
Relation  of  colloids  to,  312,  414. 
Adsorption  by,  414,  431. 
Complexes  of,  with  sodium  citrate, 

424,  429. 

Dissociation        of        agglutination 
caused  by,  313,  318,  322,  324. 


Precipitates  —  Continued. 
Serum  precipitates: 

in  anticholera  serum,  145. 

as    explaining    antagonistic    prop- 
erty of  normal  serum,  353. 

in  hemolytic  serum,  148. 

Alexin  deviation  due  to,  357. 

Action  of,  on  sensitizing  substance, 
349. 

Alexin  fixation  by,  346. 

Specificity  of,  149. 

Preventive   Serum,    (syn.    Immune    Se- 
rum) — 
Combined    action    of,    with    normal 

serum,  58,  66,  103,  135. 
Chemiotaxis  by,  52. 
Mode  of  action  of,  81. 
(see   Preventive    Substance;  Pfeiffer's 

Phenomenon). 
Preventive    Substance     (syn.    Immune 

Body;  Sensitizing  Substance)  — 
in  anticholera  serum,  51  et  seq. 
in  aqueous  humor,  73,  289. 
in  edema  fluid,  38,  73. 
Chemiotaxis  by,  52,  80. 
Effect  of  heat  on,  46,  68,  88,  134. 
Effect  of,  on  bacteria,  90,  225. 
Effect  of  injection  of,  79. 
Origin  of,  in  serum,  54,  162. 
Relation  of  leucocytes  to,  38,  41. 
Relation  of,  to  agglutinins,  97. 
Specificity  of,  48,  60,  78. 

Bat  Serum  — 

Hemagglutinins  in,  92,  178. 

Effect  of  heated,  on  B.  anthracis,  181. 
Babbit  Serum  — 

Antagonistic  effect  of,  351,  400. 

Bactericidal  action  of,  26. 

Hemagglutinins  of,  92,  178. 

Hemolysins  of,  179,  367. 

Precipitins  for,  249. 

Reactivation  of  antirabbit  serum  by, 
138,  188,  333,  334. 

Sensitizers  for,  249. 

Specific  agglutinins  in,  182. 
Beceptors  (see  Ehrlich's  Theory),  363. 

Antisensitizers  as  solution  of,  297. 

Relation   of,    to   formation   of   anti- 
bodies, 296,  301,  305,  497. 
Bed  Blood  Corpuscles  — 

Agglutination   of,  see  Hemagglutina- 
tion. 

Agglutination  of,   by  chemical   pre- 
cipitates, 312. 

Agglutination  of,  by  mastic,  432. 

Difficulty  in  washing,  347. 

Dissolution  of,  see  Hemolysis. 

Electric  charge  of,  313. 

Effect  of  agglutination  on  morphol- 
ogy of,  93. 


544 


INDEX    OF    SUBJECTS. 


Salt  Solution,   Action  of,   in  hemolytic 

experiments,    296,    301,    335,    341, 

383,  399,  451,  453,  456. 
Selection,     Effect     of     phagocytic,     in 

increasing  virulence  of  bacteria,  4, 

6,  15. 
Sensitized  Blood  Corpuscles  — 

Action  of  alexin  on,  196,  337,  365. 
Action  of  alexin  plus  conglutinin  on, 

374  et  seq. 

Action  of  opsonins  on,  395. 
Effect  of  washing  on,  172,  185. 
Action  of  antisensitizer  on,  283,  287. 
Fixation  of  alexin  on,  190,  219,  230, 

234,  244,  261,  363,  365,  398,  510. 
Suppression  of  cure  of,  by  antisen- 
sitizer, 288. 
Sensitizing  Substance,   (syn.    Preventive 

Substance;    Immune    Body;   Sensi- 

tizer;  Amboceptor). 
as  characteristic  of  specific  immune 

serum,  160,  161,  187. 
against  albuminous  substances,  241, 

246,  257,  301. 

for  bacteria  (see  under  various  Micro- 
organisms). 

for  egg  white,  248,  254. 
for  fibrinogen,  249,  255. 
for  milk,  253. 
for  serum,  241  ;   (also  under  various 

Sera). 

in  antimicrobial  sera,  217. 
in  «ormal  sera,   184,  240,  288,  372, 

379,     408,     444  ;     (see    also    under 

Hemolysins). 
Absorption  of,  by  antigen,  160,  171, 

191,  229. 
Absorption   of,    by   stromata   of   red 

blood  cells,  193. 
Action  of  antisensitizer  on,  285,  286, 

287,  513. 

Action  of  precipitates  on,  349. 
Action    of,    on    heated    blood    cells, 

337. 

Antitoxin  to  ;  (see  Antisensitizer). 
Demonstration  of,  by  fixation  reac- 
tion, 218,  245,  250,  348,  467;  (see 

also  Fixation  Reaction). 
Effect  of  dilution  on,  174. 
Effect  of  heat  on,  174,  183,  185. 
Fixation  of,  considered  as  similar  to 

a  dyeing   process,    194,   216,   230, 

262. 

Mode  of  action  of,  172,  174,  436. 
Multiplicity  of  partial,  (Ehrlich  and 

Morgenroth),  235,  302,  333. 
Relation  of  alexin  to,  172,  214,  229, 

234,  239,  334,  357,  3G3,  368,  387, 

403,  440,  511. 
Relation  of  opsonins  to,  397. 


Serum  — 

of  vaccinated  animals,  8,  56. 

Detection  of,  by  mastic,  347. 

Properties  of  specific,  159. 

(see  Normal  Serum;  Immune  Serum; 
Anticholera  Serum  ;  Antistreptococcus 
Serum ;   also   under  various   Micro- 
organisms). 
Silicic     Acid,     Agglutination    of    blood 

corpuscles  by,  313. 
Sodium  Citrate  - 

Action  of,  on  calcium  chloride,  425, 
434. 

Action  of,  due  to  acid  radicle,  424. 

Antiflocculating  action  of,  404. 

Complexes    of,    with    chemical    pre- 
cipitates, 424,  429. 

Dissemination    of    chemical    precipi- 
tates by,  424. 

Inhibiting  effect  of,  on  agglutination, 
432. 

Inhibiting  effect  of,   on  alexin  fixa- 
tion, 404,  512. 

Inhibiting    effect    of,    on    biological 

hemolysins,  404,  433. 
Stains  — 

for  bacillus  of  avian  diphtheria,  492. 

for  bacillus  of  whooping-cough,  478. 

for  Pfeiffer's  phenomenon,  63. 

for  phagocytosis,  22,  33. 

for  streptococcus  infection,  106,  123, 

129. 
Stimulin,  79. 

as  identical  with  opsonin,  389. 
Streptococcus  — 

Agglutination  of,  91,  92,  118. 

Areola  formed  about  resistant,  106, 
109. 

Culture  medium  for,  104. 

Destruction  of,  by  leucocytic  extract, 
120. 

Effect  of  age  on   virulence  of,    112, 
123  et  seq. 

Negative    chemiotaxis    by,    13,    15, 
106,  112,  113. 

Phagocytosis  of,  37. 

(sec  Antistreptococcus  Serum;  Strep- 
tococcus Infection). 
Streptococcus  Infection  — 

in  the  guinea-pig,  104,  114. 

in  the  rabbit,  109,  111. 

Delayed  phagocytosis  in,  124,  127. 

Exudate  in,  110,  124. 

Phagolysis  in,  105. 

Phagocytosis  in,  14,  108,  128,  130. 

Portal  of  entry  in,  116,  132. 

Mechanism  of  cure  in,  108. 

Relapse  or  re-infection  in,  109,  113t 
115,  129. 

Stains  for  studying,  106,  123,  129. 


INDEX    OF    SUBJECTS. 


545 


Stromata  — 

Agglutination  of,  by  chemical  pre- 
cipitates, 318. 

Fixation  of  sensitizing  substance  by, 
192,  216. 

Production  of  hemolytic  serum  by 
injection  of,  194. 

Tetanus  Bacillus,  see  B.  Tetani. 
Toxins  — 

of  symptomatic  anthrax,  520. 

Mode  of  action  of  antitoxins  on,  259, 

295,  518. 
Susceptibility   to,    in     animals     with 

lowered  resistance,  278. 
(see  Antitoxin;  Diphlheria  Toxin;  En- 

dotoxin). 
Toxoids,  279. 
Toxons  — 

Arrhenius  and  Madsen's  conception 

of,  295. 

Avidity  of,  for  antitoxin,  267. 
Bordet's  conception  of,  278,  295. 
Ehrlich's  conception  of,  267. 
Tubercle  Bacillus,  see  Bacillus  Tubercu- 


Typhold  Bacillus,  see  Bacillus  Typhosus. 
Typhoid  Fever,  Fixation  reaction  in  con- 
valescents from,  222. 

Vaccinated  Animals  — 

Edema  fluid  in,  73. 

Aqueous  humor  in,  73. 

Serum  of,  8,  56. 

Phagocytosis  in,  11. 

Bactericidal  substance  in,  45,  69,  75, 

78,  201. 

(see  also  Immunized  Animals). 
Venom,  Hemolysis  by,  367,  433,513. 
Vibrios,  Diagnosis  of,  by  Pfeiffer's  phe- 
nomenon, 44,  59,  61,  94. 
(see  V.  Choleras,  V.  Metchnikovi  and 
the  like). 


Vibrio  Cholerae,  17,  18,  22,  27,  31. 

Agglutination  of,  65,  90,  91,  96,  97, 

100,  102,  103. 

Antiserum  to,  22,  29,  44,  47,  65,  74. 
(see  also  Anticholera  Serum). 
Artificial    resistance    of,    to    aggluti- 
nation, 98. 

Diagnosis  of,  42,  59,  60,  94. 
Effects  produced  by  products  of,  51, 

92. 

Pfeiffer's  phenomenon  with,  59,  60. 
Phagocytosis  of,  2,  9,  19,  21,  22,  34, 

100. 
Vibrio  Massaouah,  21,  27. 

Antiserum  to,  21,  28,  30,  38,  43. 
Chemiotaxis  by,  52. 
Effect  of  anticholera  serum  on,  95. 
Effect    of    culture    products    of,    on 

leucocytes,  53. 

Exudate  in  infections  due  to,  40. 
Vibrio  of  Deneke,  42. 
Vibrio  Finkleri,  42. 
Vibrio  Metchnikavi,  1  et  seq.,  17. 
Antiserum  to,  44,  49. 
Pfeiffer's  phenomenon  with,  64. 
Virulence  — 

Increase  in,  of  bacteria  by  selection, 

4,  6,  15. 

Relation  of  an  areola  to,  107. 
Relation  of  negative  chemiotaxis  to, 
113. 

Whooping-cough,  407. 
Bacillus  of,  472  (q.  v.). 
Difficulty  of   isolating  specific  cause 

of,  472. 
Occurrence  of  influenza  bacillus  in, 

475,  483. 
Pathogenesis  of,  490. 

Zwlschenkbrper,  (Ehrlich  and  Morgen- 
roth)  syn.  for  sensitizing  substance 
of  normal  serum,  230,  232. 


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BRIDGES   AND    ROOFS.     HYDRAULICS.     MATERIALS   OF   ENGINEER- 
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8 


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11 


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12 


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13 


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Klein.) 8vo,  5  00 

Machinery  of  Transmission  and  Governors.      (Hermann — Klein.).  .8vo,  5  00 

Wood's  Turbines 8vo,  2  50 


MATERIALS   OF   ENGINEERING. 

*  Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  50 

Burr's  Elasticity  and  Resistance  of  the  Materials  of  Engineering 8vo,  7  50 

Church's  Mechanics  of  Engineering 8vo,  6  00 

*  Greene's  Structural  Mechanics 8vo,  2  50 

*  Holley's  Lead  and  Zinc  Pigments Large  12mo  3  00 

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Steels,  Steel-Making  Alloys  and  Graphite Large  12mo,  3  00 

Johnson's  (J.  B.)  Materials  of  Construction 8vo,  6  00 

Keep's  Cast  Iron 8vo,  2  50 

Lanza's  Applied  Mechanics 8vo,  7  50 

Maire's  Modern  Pigments  and  their  Vehicles 12mo,  2  00 

Martens's  Handbook  on  Testing  Materials.      (Henning.) 8vo;  7  50 

Maurer's  Techincal  Mechanics 8vo,  4  00 

Merriman's  Mechanics  of  Materials 8vo,  5  00 

*  Strength  of  Materials 12mo,  1  00 

Metcalf's  Steel.     A  Manual  for  Steel-users 12mo,  2  00 

Sabin's  Industrial  and  Artistic  Technology  of  Paint  and  Varnish 8vo,  3  00 

Smith's  ((A.  W.)  Materials  of  Machines 12mo,  1  00 

Smith's  (H.  E.)  Strength  of  Material 12mo. 

Thurston's  Materials  of  Engineering 3  vols.,  8vo,  8  00 

Part  I.     Non-metallic  Materials  of  Engineering, 8vo,  2  00 

Part  II.      Iron  and  Steel 8vo.  3  50 

Part  III.     A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Wood's  (De  V.)  Elements  of  Analytical  Mechanics 8vo,  3  00 

Treatise  on    the    Resistance    of    Materials    and    an    Appendix    on    the 

Preservation  of  Timber 8vo,  2  00 

Wood's  (M.  P.)  Rustless  Coatings:    Corrosion  and  Electrolysis  of  Iron  and 

Steel 8vo,  4  00 


STEAM-ENGINES    AND   BOILERS. 

Berry's  Temperature-entropy  Diagram 12mo,  2  00 

Carnot's  Reflections  on  the  Motive  Power  of  Heat.     (Thurston.) 12mo.  1  50 

Chase's  Art  of  Pattern  Making 12mo.  2  50 

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Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.  ..  .  16mo,  mor.  5  00 

Ford's  Boiler  Making  for  Boiler  Makers 18mo.  1  00 

*  Gebhardt's  Steam  Power  Plant  Engineering 8vo,  6  00 

Goss's  Locomotive  Performance Svo,  5  00 

Hemenway's  Indicator  Practice  and  Steam-engine  Economy 12mo.  2  00 

Hutton's  Heat  and  Heat-engines Svo,  5  00 

Mechanical  Engineering  of  Power  Plants Svo,  5  0( 

Kent's  Steam  boiler  Economy Svo,  4  00 

14 


Kneass's  Practice  and  Theory  of  the  Injector 8vo,  $1  50 

MacCord's  Slide-valves 8vo,  2  00 

Meyer's  Modern  Locomotire  Construction 4  to,  10  00 

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Peabody's  Manual  of  the  Steam-engine  Indicator 12mo.  1  50 

Tables  of  the  Properties  of  Steam  and  Other  Vapors  and  Temperature- 
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Valve-gears  for  Steam-engines 8vo.  2  50 

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Smart's  Handbook  of  Engineering  Laboratory  Practice 12mo,  2  50 

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Spangler's  Notes  on  Thermodynamics 12mo.  1  00 

Valve-gears 8vo,  2  50 

Spangler,  Greene,  and  Marshall's  Elements  of  Steam-engineering 8vo  3  00 

Thomas's  Steam-turbines 8vo,  4  00 

Thurston's  Handbook  of  Engine  and  Boiler  Trials,  and  the  Use  of  the  Indi- 
cator and  the  Prony  Brake 8vo,  5  00 

Handy  Tables 8vo,  1  50 

Manual  of  Steam-boilers,  their  Designs,  Construction,  and  Operation  8vo.  5  00 

Manual  of  the  Steam-engine 2vols..  8vo.  10  00 

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Part  II.      Design,  Construction,  and  Operation 8vo,  6  00 

Steam-boiler  Explosions  in  Theory  and  in  Practice 12mo,  1  50 

Wehrenfenning's  Analysis  and  Softening  of  Boiler  Feed-water.     (Patterson). 

8vo,  4  00 

Weisbach's  Heat,  Steam,  and  Steam-engines.     (Du  Bois.) 8vo.  5  00 

Whitham's  Steam-engine  Design 8vo,  5  00 

Wood's  Thermodynamics,  Heat  Motors,  and  Refrigerating  Machines.  .  .  8vo,  4  00 


MECHANICS    PURE   AND    APPLIED. 

Church's  Mechanics  of  Engineering 8vo,  6  00 

Notes  and  Examples  in  Mechanics 8vo.  2  00 

Dana's  Text-book  of  Elementary  Mechanics  for  Colleges  and  Schools  .12mo,  1  50 
Du  Bois's  Elementary  Principles  of  Mechanics: 

Vol.    I.     Kinematics 8vo.  3  50 

Vol.  II.     Statics 8vo,  4  00 

Mechanics  of  Engineering.     Vol.    I Small  4to,  7  50 

Vol.  II Small  4to,  10  00 

*  Greene's  Structural  Mechanics 8vo,  2  50 

James's  Kinematics  of  a  Point  and  the  Rational  Mechanics  of  a  Particle. 

Large  12mo,  2  00 

*  Johnson's  (W.  W.)  Theoretical  Mechanics 12mo,  3  00 

Lanza's  Applied  Mechanics 8vo.  7  50 

*  Martin's  Text  Book  on  Mechanics.  Vol.  I,  Statics 12mo,  1  25 

*  Vol.  II,  Kinematics  and  Kinetics.  12mo.  1   50 

Maurer's  Technical  Mechanics 8vo.  4  00 

*  Merriman's  Elements  of  Mechanics 12mo,  1  00 

Mechanics  of  Materials 8vo,  5  00 

*  Michie's  Elements  of  Analytical  Mechanics 8vo,  4  00 

Robinson's  Principles  of  Mechanism 8vo,  3  00 

Sanborn's  Mechanics  Problems Large  12mo,  1   50 

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15 


MEDICAL. 

*  Abderhalden's  Physiological  Chemistry  in   Thirty  Lectures.     (Hall  and 

Defren.) 8vo,  $5  00 

von  Behring's  Suppression  of  Tuberculosis.      (Bolduan.) 12mo,  1  00 

Bolduan's  Immune  Sera 12mo,  1  50 

Bordet's  Studies  in  Immunity.      (Gay).      (In  Press.) 8vo, 

Davenport's  Statistical  Methods  with  Special  Reference  to  Biological  Varia- 
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Ehrlich's  Collected  Studies  on  Immunity.      (Bolduan.) 8vo,  6  00 

*  Fischer's  Physiology  of  Alimentation Large  12mo,  2  00 

de  Fursac's  Manual  of  Psychiatry.      (Rosanoff  and  Collins.)..  .  .Large  12mo,  2  50 

Hammarsten's  Text-book  on  Physiological  Chemistry.      (Mandel.) 8vo,  4  00 


Jackson's  Directions  for  Laboratory  Work  in  Physiological  Chemistry.  .8vo, 

Lassar-Cohn's  Practical  Urinary  Analysis.      (Lorenz.) 12mo, 

Mandel's  Hand-book  for  the  Bio-Chemical  Laboratory 12mo, 

*  Pauli's  Physical  Chemistry  in  the  Service  of  Medicine.      (Fischer.)  ..12mo, 

*  Pozzi-Escot's  Toxins  and  Venoms  and  their  Antibodies.      (Cohn.).  .  12mo, 


25 
00 
50 
25 
00 
00 


Rostoski's  Serum  Diagnosis.      (Bolduan.) 12mo, 

Ruddiman's  Incompatibilities  in  Prescriptions 8vo,  2  00 

Whys  in  Pharmacy 12mo,  1  00 

Salkowski's  Physiological  and  Pathological  Chemistry.     (Orndorff.)  ....8vo,  2  50 

*  Satterlee's  Outlines  of  Human  Embryology 12mo,  1  25 

Smith's  Lecture  Notes  on  Chemistry  for  Dental  Students 8vo,  2  50 

*  Whipple's  Tyhpoid  Fever Large  12mo,  3  00 

Woodhull's  Notes  on  Military  Hygiene 16mo,  1  50 

*  Personal  Hygiene 12mo,  1  00 

Worcester  and  Atkinson's  Small  Hospitals  Establishment  and  Maintenance, 
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METALLURGY. 

Betts's  Lead  Refining  by  Electrolysis 8vo,  4  00 

Bolland's  Encyclopedia  of  Founding  and  Dictionary  of  Foundry  Terms  used 

in  the  Practice  of  Moulding 12mo,  3  00 

Iron  Founder 12mo,  2  50 

Supplement 12mo,  2  50 

Douglas's  Untechnical  Addresses  on  Technical  Subjects 12mo,  1  00 

Goesel's  Minerals  and  Metals:  A  Reference  Book 16mo,  mor.  3  00 

*  Iles's  Lead-smelting 12mo,  2  50 

Johnson's    Rapid    Methods   for    the   Chemical   Analysis   of   Special   Steels, 

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Keep's  Cast  Iron 8vo,  2  50 

Le  Chatelier's  High- temperature  Measurements.     (Boudouard — Burgess.) 

12mo,  3  00 

Metcalf 's  Steel.     A  Manual  for  Steel-users 12mo.  2  00 

Minet's  Production  of  Aluminum  and  its  Industrial  Use.     (Waldo.).  .  12mo,  2  50 

Ruer's  Elements  of  Metallography.      (Mathewson) 8vo. 

Smith's  Materials  of  Machines 12mo,  1  00 

Tate  and  Stone's  Foundry  Practice 12mo,  2  00 

Thurston's  Materials  of  Engineering.     In  Three  Parts 8vo,  8  00 

Part  I.       Non-metallic  Materials  of  Engineering,  see  Civil  Engineering, 
page  9. 

Part  II.     Iron  and  Steel 8vo,  3  50 

Part  III.  A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Ulke's  Modern  Electrolytic  Copper  Refining 8vo,  3  00 

West's  American  Foundry  Practice 12mo,  2  50 

Moulders'  Text  Book 12mo,  2  50 

16 


MINERALOGY. 

Baskerville's  Chemical  Elements.     (In  Preparation.). 

Boyd's  Map  of  Southwest  Virginia Pocket-book  form.  $2  00 

*  Browning's  Introduction  to  the  Rarer  Elements 8vo,  1   50 

Brush's  Manual  of  Determinative  Mineralogy.      (Penfield.) 8vo,  4  00 

Butler's  Pocket  Hand-book  of  Minerals 16mo,  mor.  3  00 

Chester's  Catalogue  of  Minerals 8vo,  paper,     1  00 

Cloth,  1  25 

*  Crane's  Gold  and  Silver 8vo,  5  00 

Dana's  First  Appendix  to  Dana's  New  "System  of  Mineralogy".  .Large  8vo,  1  00 
Dana's  Second  Appendix  to  Dana's  New  "  System  of  Mineralogy." 

Large  8vo, 

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Minerals  and  How  to  Study  Them 12mo,  1  50 

System  of  Mineralogy Large  8vo,  half  leather,  12  50 

Text-book  of  Mineralogy 8vo,  4  00 

Douglas's  Untechnical  Addresses  on  Technical  Subjects 12mo,  1  00 

Eakle's  Mineral  Tables 8vo,  1  25 

Eckel's  Stone  and  Clay  Products  Used  in  Engineering.      (In  Preparation). 

Goesel's  Minerals  and  Metals:  A  Reference  Book 16mo,  mor.  3  00 

Groth's  Introduction  to  Chemical  Crystallography  (Marshall) 12mo,  1  25 

*  Hayes's  Handbook  for  Field  Geologists 16mo,  mor.  1  50 

Iddings's  Igneous  Rocks 8vo,  5  00 

Rock  Minerals 8vo,  5  00 

Johannsen's  Determination  of  Rock-forming  Minerals  in  Thin  Sections.  8vo, 

With  Thumb  Index  5  00 

*  Martin's  Laboratory     Guide    to    Qualitative    Analysis    with    the    Blow- 

pipe  12mo,  60 

Merrill's  Non-metallic  Minerals:  Their  Occurrence  and  Uses 8vo,  4  00 

Stones  for  Building  and  Decoration 8vo,  5  00 

*  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

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Tables  of   Minerals,    Including  the  Use  of  Minerals  and   Statistics  of 

Domestic  Production 8vo,  1  00 

*  Pirsson's  Rocks  and  Rock  Minerals 12mo,  2  50 

*  Richards's  Synopsis  of  Mineral  Characters 12mo,  mor.  1  25 

*  Ries's  Clays :  Their  Occurrence,  Properties  and  Uses 8vo,  5  00 

*  Ries  and  Leighton's  History  of  the  Clay-working  Industry  of  the  United 

States 8vo,  2  50 

*  Tillman's  Text-book  of  Important  Minerals  and  Rocks 8vo,  2  00 

Washington's  Manual  of  the  Chemical  Analysis  of  Rocks , 8vo,  2  00 


MINING. 

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*  Crane's  Gold  and  Silver 8vo,  5  00 

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*  8vo,  mor.  5  00 

Douglas's  Untechnical  Addresses  on  Technical  Subjects 12mo,  1   00 

Eissler's  Modern  High  Explosives 8vo,  4  00 

Goesel's  Minerals  and  Metals:  A  Reference  Book 16mo,  mor.  3  00 

Ihlseng's  Manual  of  Mining 8vo,  5  00 

*  Iles's  Lead  Smelting 12mo,  2  50 

Peele's  Compressed  Air  Plant  for  Mines 8vo,  3  00 

Riemer's  Shaft  Sinking  Under  Difficult  Conditions.     (Corning  and  Peele).8vo,  3  00 

*  Weaver's  Military  Explosives 8vo,  3  00 

Wilson's  Hydraulic  and  Placer  Mining.      2d  edition,  rewritten 12mo,  2  50 

Treatise  on  Practical  and  Theoretical  Mine  Ventilation 12mo,  1  25 

17 


SANITARY   SCIENCE. 

Association  of  State  and  National  Food  and  Dairy  Departments,  Hartford 

Meeting,  1906 8vo,  $3  00 

Jamestown  Meeting,  1907 8vo,  3  00 

*  Bashore's  Outlines  of  Practical  Sanitation 12mo,  1  25 

Sanitation  of  a  Country  House 12mo,  1  00 

Sanitation  of  Recreation  Camps  and  Parks 12mo,  1  00 

Folwell's  Sewerage.      (Designing,  Construction,  and  Maintenance.) 8vo,  3  00 

Water-supply  Engineering 8vo,  4  00 

Fowler's  Sewage  Works  Analyses 12mo,  2  00 

Fuertes's  Water-filtration  Works 12mo,  2  50 

Water  and  Public  Health 12mo,  1  50 

Gerhard's  Guide  to  Sanitary  Inspections 12mo,  1  50 

*  Modern  Baths  and  Bath  Houses 8vo,  3  00 

Sanitation  of  Public  Buildings 12mo,  1  50 

Hazen's  Clean  Water  and  How  to  Get  It Large  12mo,  1  50 

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Mason's  Examination  of  Water.      (Chemical  and  Bacteriological) 12mo,  1  25 

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*  Merriman's  Elements  of  Sanitary  Enigneering 8vo,  2  00 

Ogden's  Sewer  Design 12mo,  2  00 

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Prescott  and  Winslow's  Elements  of  Water  Bacteriology,  with  Special  Refer- 
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Richards's  Cost  of  Cleanness 12mo,  1  00 

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International  Congress  of  Geologists Large  8ro.  1  50 

Ferrel's  Popular  Treatise  on  the  Winds 8vo,  4  00 

Fitzgerald's  Boston  Machinist 18mo,  1  00 

Gannett's  Statistical  Abstract  of  the  World 24mo,  75 

Haines's  American  Railway  Management 12mo,  2  50 

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18 


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Metcalfe's  Cost  of  Manufactures,  and  the  Administration  of  Workshops.. 8vo,  5  00 

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Ricketts's  History  of  Rensselaer  Polytechnic  Institute  1824-1894. 

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Thome's  Structural  and  Physiological  Botany.      (Bennett) 16mo,  2  25 

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Winslow's  Elements  of  Applied  Microscopy 12mo,  1  50 


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Gesenius's  Hebrew  and  Chaldee  Lexicon  to  the  Old  Testament  Scriptures. 

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QR181  Bordet,    Jules,          D1330 

B72  Studies  in  immunity , by 

1909  Professor  Jules  Bordet~.  an| 

c,2  hils  collaborators.   CO'JL-L. 


1st  ed« 


ICT  13  1! 


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